NIR-II Fluorescence Imaging: Revolutionizing Deep-Tissue Visualization for Biomedical Research and Clinical Translation

Benjamin Bennett Nov 29, 2025 231

This article comprehensively explores second near-infrared (NIR-II, 1000–1700 nm) fluorescence imaging, an advanced optical modality offering superior tissue penetration and high-fidelity visualization for preclinical research and emerging clinical applications.

NIR-II Fluorescence Imaging: Revolutionizing Deep-Tissue Visualization for Biomedical Research and Clinical Translation

Abstract

This article comprehensively explores second near-infrared (NIR-II, 1000–1700 nm) fluorescence imaging, an advanced optical modality offering superior tissue penetration and high-fidelity visualization for preclinical research and emerging clinical applications. We detail the fundamental principles that enable NIR-II light to minimize photon scattering, reduce tissue autofluorescence, and achieve a high signal-to-background ratio, surpassing the limitations of visible and NIR-I imaging. The scope encompasses the latest methodological advances, including a diverse palette of organic and inorganic fluorophores, state-of-the-art imaging systems, and their application in vascular mapping, cancer surgery navigation, and real-time therapeutic monitoring. We further address critical challenges in fluorophore optimization and biological barriers, provide a comparative analysis with established imaging techniques, and discuss the promising pathway toward clinical adoption, providing researchers and drug development professionals with a vital resource in this cutting-edge field.

Unlocking the NIR-II Window: Fundamental Principles and Superior Advantages for Deep-Tissue Imaging

The interaction of light with biological tissue is governed by three fundamental physical phenomena: scattering, absorption, and autofluorescence. These properties collectively determine the penetration depth, spatial resolution, and signal-to-background ratio (SBR) achievable in biomedical optical imaging [1] [2]. When photons enter tissue, they may be absorbed by chromophores such as hemoglobin, lipids, melanin, and water, with their energy converted to heat or re-emitted as fluorescence. Alternatively, photons may be scattered by microscopic variations in refractive index within cellular and extracellular structures, changing their direction of propagation without energy loss. Autofluorescence originates from endogenous fluorophores including collagen, elastin, and flavins, generating background signal that can obscure specific contrast agent signals [3] [4].

The second near-infrared window (NIR-II, 900-1700 nm) has emerged as a superior regime for deep-tissue fluorescence imaging compared to the traditional first near-infrared window (NIR-I, 700-900 nm) and visible spectrum (400-700 nm) [1] [3] [5]. This advantage stems from significantly reduced scattering coefficients, diminished autofluorescence, and lower absorption by dominant tissue chromophores in this spectral region. Consequently, NIR-II fluorescence imaging enables deeper tissue penetration (5-20 mm) and higher spatio-temporal resolution, making it particularly valuable for pre-clinical research and clinical translation [1] [4].

Table 1: Comparison of Optical Windows for Biomedical Imaging

Optical Property Visible (400-700 nm) NIR-I (700-900 nm) NIR-II (900-1700 nm)
Tissue Scattering High Moderate Low
Tissue Autofluorescence High Moderate Low
Absorption by Hemoglobin High Moderate Low
Absorption by Water Low Low Moderate (increases with wavelength)
Penetration Depth Shallow (1-2 mm) Moderate (2-5 mm) Deep (5-20 mm)
Spatial Resolution Low Moderate High

Fundamental Physics of Light-Tissue Interactions

Photon Absorption in Biological Tissues

Absorption in biological tissues follows the Beer-Lambert law, where the probability of photon absorption depends on both the concentration of chromophores and their wavelength-dependent extinction coefficients. Principal absorbers in the NIR region include hemoglobin (oxy- and deoxy-forms), lipids, melanin, and water [6] [5]. While absorption is traditionally viewed as detrimental to signal detection, moderate absorption can paradoxically enhance image quality by preferentially attenuating multiply-scattered photons that contribute to background noise [5]. This phenomenon creates an absorption-based gating effect that improves spatial resolution and SBR by suppressing longer-path-length photons [5].

The absorption spectrum of water, the dominant tissue component, reveals several peaks in the NIR region at approximately 980 nm, 1200 nm, 1450 nm, and 1930 nm [5]. Regions surrounding these peaks (900-1000 nm, 1100-1250 nm, 1300-1500 nm, and 1700-1880 nm) benefit from this absorption-mediated background suppression, enabling high-fidelity imaging despite increased signal attenuation [5].

Photon Scattering in Turbid Biological Media

Scattering events in tissues arise from refractive index mismatches at cellular and subcellular interfaces, including membranes, organelles, and collagen fibrils. The scattering coefficient (μs) and anisotropy factor (g) collectively determine the reduced scattering coefficient (μs' = μs(1-g)), which decreases with increasing wavelength according to an approximate power law relationship [5]. This wavelength dependence means that NIR-II photons experience significantly less scattering than visible or NIR-I photons, enabling better preservation of ballistic and snake photons that carry spatial information [1] [4].

Reduced scattering in the NIR-II window allows light to be focused more tightly and deeply within tissue, dramatically improving spatial resolution for microscopic applications. The point spread function becomes narrower, and the degree of light diffusion decreases, permitting high-resolution visualization of subcellular structures and microvascular networks at unprecedented depths [4] [5].

Tissue Autofluorescence Origins and Characteristics

Autofluorescence originates from endogenous fluorophores including collagen, elastin, flavins, lipofuscin, and NAD(P)H [3]. These compounds typically exhibit excitation and emission in the visible spectrum, with minimal emission beyond 900 nm. This property creates an exceptionally low autofluorescence background in the NIR-II window, resulting in significantly higher SBR compared to visible and NIR-I imaging [1] [4]. The combination of reduced scattering, favorable absorption profiles, and diminished autofluorescence in the NIR-II window enables high-contrast imaging of deep-tissue structures with micron-scale resolution [3] [5].

Table 2: Key Optical Properties Across the Near-Infrared Spectrum

Wavelength Range Reduced Scattering Coefficient (μs') Absorption Coefficient (μa) of Water Autofluorescence Intensity Recommended Applications
NIR-I (700-900 nm) ~1.0 mm⁻¹ Low Moderate Superficial vascular imaging, lymph node mapping
NIR-IIa (900-1300 nm) ~0.5 mm⁻¹ Low to Moderate Low Deep-tissue vascular imaging, tumor detection
NIR-IIb (1500-1700 nm) ~0.3 mm⁻¹ Moderate Very Low High-resolution microscopic imaging
NIR-IIx (1400-1500 nm) ~0.3 mm⁻¹ High (near water peak) Very Low Maximum contrast for bright probes
NIR-IIc (1700-1880 nm) ~0.2 mm⁻¹ Moderate to High Negligible Emerging applications with new detectors

Optimized NIR-II Imaging Windows

Theoretical Basis for Window Definition

Monte Carlo simulations of photon transport in biological media have refined our understanding of optimal imaging windows within the NIR-II spectrum [5]. These simulations demonstrate that while scattering decreases monotonically with increasing wavelength, strategic utilization of water absorption peaks can further enhance image quality by preferentially suppressing multiply-scattered background photons [5]. This counterintuitive principle has led to the identification of several sub-windows within the broader NIR-II region that offer exceptional imaging performance.

The NIR-IIx window (1400-1500 nm) and NIR-IIc window (1700-1880 nm) are located near water absorption peaks and provide superior image contrast compared to adjacent regions despite higher absolute absorption [5]. Beyond 1880 nm, the NIR-III window (2080-2340 nm) has been proposed as potentially offering the ultimate imaging quality due to minimal scattering, though current detector technology limits its practical implementation [5].

NIRWindows NIR_I NIR-I Window 700-900 nm NIR_II NIR-II Window 900-1880 nm NIR_I->NIR_II NIR_IIa NIR-IIa 900-1300 nm NIR_II->NIR_IIa NIR_IIb NIR-IIb 1500-1700 nm NIR_II->NIR_IIb NIR_IIx NIR-IIx 1400-1500 nm NIR_II->NIR_IIx NIR_IIc NIR-IIc 1700-1880 nm NIR_II->NIR_IIc NIR_III NIR-III Window 2080-2340 nm NIR_II->NIR_III

Diagram 1: Hierarchical classification of near-infrared imaging windows, showing the relationship between major windows and sub-windows with their respective wavelength ranges.

Quantitative Performance Metrics Across Windows

The performance advantages of NIR-II imaging can be quantified through several key metrics. Spatial resolution in scattering media improves with increasing wavelength due to reduced scattering, with NIR-II imaging demonstrating approximately 2-3 times higher resolution than NIR-I imaging at equivalent depths [1] [5]. The signal-to-background ratio (SBR) benefits from both reduced autofluorescence and absorption-mediated suppression of multiply-scattered photons, with NIR-II imaging achieving 5-10 times higher SBR compared to NIR-I imaging [1] [4]. Penetration depth increases to 5-20 mm in the NIR-II window, enabling non-invasive visualization of deep-tissue structures in small animal models and potentially in clinical applications [1] [3].

For fluorescence microscopy applications, NIR-II wide-field microscopy in the brain has demonstrated exceptional optical sectioning capability with imaging depths reaching ~1.3 mm through intact skull, representing the deepest in vivo NIR-II fluorescence microscopy achieved to date [5]. This performance underscores the transformative potential of optimized window selection for advanced imaging applications.

Research Reagent Solutions for NIR-II Imaging

Table 3: Key Research Reagents for NIR-II Fluorescence Imaging

Reagent Category Specific Examples Key Properties Primary Applications
Inorganic Nanomaterials PbS/CdS QDs [5], Rare-earth doped nanoparticles (RENPs) [1] [3], Carbon nanotubes (CNTs) [1] High quantum yield, narrow emission, tunable wavelengths, excellent photostability Multiplexed imaging, deep-tissue angiography, cellular tracking
Organic Small Molecules CH1055 [3], FEB dyes [3], IR-BEMC6P [3], D-A-D structured dyes [1] [3] Well-defined structures, renal clearance, modifiable targeting, tunable optical properties Molecular imaging, tumor detection, pharmacokinetic studies
Activated Probes APNO-1080 (NO sensing) [6], Enzyme-activated probes [7] Low background, target-activated fluorescence, high specificity Pathological biomarker detection, enzymatic activity monitoring, tumor microenvironment sensing
Protein-Hybrid Systems HSA@CO-1080 [8], Biomimetic fluorescent proteins [8] Enhanced brightness and photostability, improved biocompatibility, covalent binding Lymphography, angiography, long-term circulation studies

Experimental Protocols for NIR-II Bioimaging

Protocol: NIR-II Fluorescence Microscopy of Cerebral Vasculature

This protocol describes wide-field fluorescence microscopy in the NIR-IIx window (1400-1500 nm) for high-resolution imaging of cerebral vasculature through intact skull, achieving exceptional penetration depth and minimal background [5].

Materials Required:

  • Bright NIR-II fluorophore (e.g., PEGylated PbS/CdS CSQDs with ~1450 nm emission) [5]
  • Animal model (e.g., mouse) with cranium exposed
  • 1064 nm laser excitation source
  • InGaAs camera with 1400 nm long-pass filter
  • Stereotactic fixation apparatus
  • Physiological monitoring equipment (body temperature, respiration)

Procedure:

  • Nanoparticle Preparation: Synthesize PbS/CdS core-shell quantum dots with emission peak at ~1450 nm. PEGylate for hydrophilicity and biocompatibility. Characterize absorption/emission spectra and quantum yield [5].
  • Animal Preparation: Anesthetize mouse and secure in stereotactic frame. Maintain body temperature at 37°C throughout procedure. Administer fluorophore via intravenous injection (dose: ~100-200 μL of 100-500 nM QD solution) [5].
  • Microscope Configuration:
    • Align 1064 nm laser for wide-field epi-illumination
    • Position InGaAs camera with 1400 nm long-pass filter
    • Optimize focus through skull surface
  • Image Acquisition:
    • Acquire time-series images at 50-100 ms exposure per frame
    • Maintain laser power density below tissue damage threshold (typically <100 mW/cm²)
  • Data Analysis:
    • Calculate vessel diameter and blood flow velocity from temporal dynamics
    • Generate maximum intensity projections for structural analysis
    • Quantify signal-to-background ratios across different cortical regions

Technical Notes: This method achieves ~1.3 mm penetration depth in mouse brain with excellent spatial resolution. The 1400-1500 nm window provides superior performance compared to NIR-IIb imaging due to optimal balance of scattering reduction and absorption-mediated background suppression [5].

Protocol: Ultrasound-Switchable Fluorescence (USF) Imaging

This advanced technique combines ultrasound focusing with NIR fluorescence to achieve spatial resolution beyond the acoustic diffraction limit in deep tissue [9].

Materials Required:

  • Thermo-sensitive USF contrast agents (ICG-encapsulated PNIPAM nanoparticles) [9]
  • High-intensity focused ultrasound (HIFU) transducer (e.g., 2.5 MHz)
  • NIR excitation source (780 nm laser)
  • Fluorescence collection system with appropriate filters
  • Tissue phantom or prepared tissue sample

Procedure:

  • Contrast Agent Preparation: Synthesize ICG-encapsulated thermo-sensitive nanoparticles with sharp transition at desired temperature (e.g., 31°C for PNIPAM NPs) [9].
  • Sample Preparation: Embed silicone tube filled with USF contrast agents in tissue phantom or porcine muscle tissue (8-10 mm thickness) to simulate blood vessel [9].
  • System Configuration:
    • Position HIFU transducer to focus on target region
    • Align excitation laser and fluorescence collection fibers
    • Submerge transducer and tissue bottom in water for acoustic coupling
  • USF Imaging:
    • Apply HIFU pulses to locally heat focal volume above LCST
    • Acquire fluorescence signal only during HIFU activation
    • Scan HIFU focus in x-y plane to reconstruct 2D image
  • Image Reconstruction:
    • Correlate fluorescence signals with HIFU focal position
    • Apply background subtraction from OFF-state fluorescence
    • Reconstruct super-resolution image

Technical Notes: USF imaging achieves spatial resolution beyond the acoustic diffraction limit (potentially <100 μm) at depths of ~8 mm in tissue. The ION/IOFF ratio of optimal contrast agents reaches 9.1, enabling excellent background suppression [9].

USFProtocol NP Synthesize thermo-sensitive nanoparticles with ICG Sample Prepare tissue sample with target structure NP->Sample Setup Configure USF system: HIFU transducer + optical path Sample->Setup HIFU Apply focused ultrasound to switch on fluorescence Setup->HIFU Detect Detect NIR fluorescence only during HIFU pulses HIFU->Detect Scan Scan HIFU focus across sample plane Detect->Scan Reconstruct Reconstruct super-resolution image from signals Scan->Reconstruct

Diagram 2: Experimental workflow for ultrasound-switchable fluorescence (USF) imaging, showing the sequence from sample preparation to super-resolution image reconstruction.

Advanced Applications and Future Perspectives

The unique advantages of NIR-II imaging have enabled diverse applications in biomedical research. Multiplexed imaging leverages multiple spectrally distinct NIR-II probes to simultaneously visualize different molecular targets or biological processes [1]. This approach enables researchers to study complex cellular interactions and disease mechanisms with unprecedented detail. Activatable molecular probes that remain dark until specifically triggered by disease biomarkers (e.g., enzymes, reactive oxygen species, or abnormal pH) provide exceptionally high target-to-background ratios for precise disease detection [7].

Emerging technologies continue to push the boundaries of NIR-II imaging. Bioluminescence resonance energy transfer (BRET)-based NIR-II systems eliminate the need for external excitation, further reducing background and enabling ultra-sensitive imaging [1]. Hybrid approaches that combine NIR-II fluorescence with other modalities like photoacoustic imaging [6] or ultrasound [9] provide complementary information for comprehensive tissue characterization.

The future clinical translation of NIR-II imaging will depend on developing fluorophores with optimal combinations of brightness, biocompatibility, and clearance properties [4] [8]. Refined imaging window definitions and the development of specialized optical systems will further enhance performance, potentially revolutionizing diagnostic imaging and therapeutic monitoring in clinical practice.

Fluorescence imaging in the second near-infrared window (NIR-II) has emerged as a transformative modality for in vivo biomedical research, enabling unprecedented capabilities for deep-tissue visualization at high spatial and temporal resolution. This imaging window fundamentally leverages favorable light-tissue interactions within the 1000-1700 nm spectrum, where reduced photon scattering, minimal tissue autofluorescence, and lower photon absorption by biological components occur compared to traditional visible (400-700 nm) and first near-infrared (NIR-I, 700-900 nm) windows [10]. The superior imaging performance within the NIR-II window provides researchers with powerful tools for investigating physiological and pathological processes in live subjects, facilitating applications ranging from vascular imaging and cancer detection to intraoperative guidance and therapeutic monitoring.

The development of NIR-II imaging represents a continuous pursuit of optimal wavelength ranges for probing biological systems. While conventional NIR-I imaging using FDA-approved dyes like indocyanine green (ICG) has provided clinical value for decades, it faces inherent limitations in penetration depth and spatial resolution due to greater tissue scattering and autofluorescence [11] [10]. The extension into NIR-II wavelengths has demonstrated remarkable improvements, achieving approximately 10-fold higher spatial resolution and significantly deeper tissue penetration up to several centimeters, making it particularly valuable for preclinical research and emerging clinical applications [12] [10].

Defining the NIR-II Spectrum and Sub-Regions

Primary NIR-II Window and Standard Subdivisions

The NIR-II window is formally defined as the spectral range from 1000 nm to 1700 nm [10] [13]. Within this primary window, researchers have established further subdivisions to categorize regions with distinct optical properties and imaging characteristics:

Table 1: Standard NIR-II Window Sub-Regions

Spectral Region Wavelength Range (nm) Key Characteristics
NIR-II 1000-1700 Primary second near-infrared window
NIR-IIa 1300-1400 Reduced scattering versus NIR-II [14]
NIR-IIb 1500-1700 Minimal tissue autofluorescence and scattering [13]

The NIR-IIb sub-region (1500-1700 nm) has received particular attention due to significantly reduced scattering and virtually negligible tissue autofluorescence, which enables superior image contrast compared to shorter wavelengths [15] [13]. However, this advantage must be balanced against increased water absorption in this region, which can attenuate fluorescence signals.

Extended and Proposed NIR-II Classifications

Recent research has further refined the NIR-II window definition and proposed extensions based on detailed analysis of light-tissue interactions:

Table 2: Extended and Proposed NIR-II Classifications

Spectral Region Wavelength Range (nm) Basis/Rationale
NIR-II (Perfected) 900-1880 Incorporates regions around water absorption peaks [13]
NIR-IIx 1400-1500 Region around water absorption peak at ~1450 nm [13]
NIR-IIc 1700-1880 Comparable properties to NIR-IIb [13]
NIR-III 2080-2340 Newly proposed window with potential for superior imaging [13]

The concept of a "perfected" NIR-II window from 900-1880 nm acknowledges the constructive role of moderate water absorption in enhancing image quality by preferentially attenuating multiply scattered photons that contribute to background noise [13]. The proposal of the NIR-III window (2080-2340 nm) suggests future directions for optical imaging, though current detector technology primarily limits practical implementation to wavelengths below 1700 nm.

Experimental Protocols for NIR-II Imaging

Protocol: Multi-Wavelength LED Excited NIR-II Imaging

This protocol describes methodology for conducting NIR-II fluorescence imaging using multi-wavelength LED excitation sources, based on recent research demonstrating the viability of LEDs as alternatives to laser-based systems [16].

Materials and Equipment
  • NIR-II Fluorophore: TPA-TQT@BSA-H complex (or alternative blue-shifted NIR-II dye)
  • Excitation Source: Multi-wavelength LED (e.g., X-Cite 110LED with peaks at 385/30 nm, 475/40 nm, 560/80 nm, and 640/40 nm)
  • Detection System: InGaAs camera with sensitivity in 1000-1700 nm range
  • Optical Filters: 1000 nm long-pass filter for emission collection
  • Animal Model: Mouse (or other appropriate animal model)
  • Anesthesia System: Isoflurane vaporizer with induction chamber
  • Image Analysis Software: NIR-II compatible analysis package
Procedure

  • Fluorophore Preparation:

    • Complex TPA-TQT dye with bovine serum albumin (BSA) to form TPA-TQT@BSA-H complex
    • Confirm complex formation and measure concentration spectrophotometrically
    • Prepare injection solution in sterile saline at appropriate concentration (typically 100-200 μM)

  • System Calibration:

    • Power on LED excitation source and allow 15 minutes for intensity stabilization
    • Configure InGaAs camera settings: set exposure time (typically 100-1000 ms), frame rate, and temperature cooling
    • Verify proper alignment of 1000 nm long-pass filter in emission pathway
    • Perform flat-field correction using standardized reflectance sample

  • Animal Preparation:

    • Anesthetize mouse using isoflurane (3-4% for induction, 1-2% for maintenance)
    • Place animal in prone position on warmed imaging stage
    • Administer fluorophore via tail vein injection (dose: 100-200 μL of prepared solution)
    • Allow 5-10 minutes for systemic circulation

  • Image Acquisition:

    • Position animal under LED excitation source
    • Set LED intensity to appropriate level (0.35 mW cm⁻² for low-power imaging)
    • Acquire time-series images at desired intervals for kinetic studies
    • For vascular imaging, acquire rapid sequence images (1-5 frames per second)
    • For tumor delineation, acquire high-resolution static images with longer exposure

  • Image Processing and Analysis:

    • Apply background subtraction using reference region without fluorescence
    • Calculate signal-to-background ratios (SBR) for regions of interest
    • For vascular imaging, measure vessel diameter and blood flow velocity
    • For tumor imaging, quantify tumor-to-normal tissue ratio
Technical Notes
  • Multi-wavelength LED excitation provides lower background compared to single-wavelength LEDs
  • This system achieves high-resolution imaging of blood vessels (∼103 μm) and lymphatic capillaries (∼129.8 μm)
  • White LED excitation causes minimal photobleaching compared to 808 nm laser irradiation
  • Optimal results obtained with blue-shifted NIR-II dyes whose absorption spectra overlap with LED emission peaks

Protocol: NIR-IIb Imaging with Off-Peak Detection of NIR-I Dyes

This protocol describes methodology for implementing NIR-IIb imaging using the long emission tails of commercially available NIR-I dyes, providing an accessible entry point to NIR-II imaging [11] [13].

Materials and Equipment
  • Fluorophores: ICG (indocyanine green) or IRDye800CW
  • Excitation Source: 808 nm laser diode
  • Detection System: InGaAs camera with extended sensitivity to 1700 nm
  • Optical Filters: 1500 nm long-pass filter for NIR-IIb imaging
  • Spectrophotometer: For verifying dye concentration and quality
Procedure

  • Dye Preparation:

    • Prepare ICG solution following manufacturer instructions
    • Protect from light to prevent photodegradation
    • Confirm concentration using absorbance at 780 nm (extinction coefficient ≈ 130,000 M⁻¹cm⁻¹)

  • System Configuration:

    • Align 808 nm laser excitation source with appropriate beam shaping
    • Install 1500 nm long-pass filter in emission path
    • Configure InGaAs camera for detection in 1500-1700 nm range
    • Validate system sensitivity using reference fluorophore

  • In Vivo Imaging:

    • Administer ICG via intravenous injection (dose: 0.1-0.3 mg/kg for vascular imaging)
    • Acquire image sequences over time to capture pharmacokinetics
    • For optimal NIR-IIb detection, use longer exposure times than NIR-I imaging

  • Data Analysis:

    • Quantify signals specifically in the 1500-1700 nm range
    • Compare image quality with simultaneous NIR-I detection
    • Calculate improvement in signal-to-background ratio relative to NIR-I imaging
Technical Notes
  • ICG exhibits non-negligible emission tail extending past 1500 nm despite peak emission at ∼830 nm
  • NIR-IIb imaging with ICG provides 2-4 fold higher tumor-to-normal tissue signal ratios compared to NIR-I imaging
  • This approach leverages clinically approved dyes, facilitating translational research
  • Detection in the NIR-IIb window significantly reduces tissue autofluorescence

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for NIR-II Imaging

Reagent/Material Function/Application Examples/Notes
Organic NIR-II Fluorophores Small-molecule fluorescent probes TPA-TQT [16], CH1055-PEG [11], IR-FGP [11], HQL2 AIE dots [15]
NIR-I Dyes with NIR-II Tails Clinically relevant probes for NIR-II imaging Indocyanine Green (ICG) [11], IRDye800CW [11]
Nanoparticle Fluorophores Inorganic probes with tunable emission PbS/CdS Quantum Dots [13], Single-Walled Carbon Nanotubes (SWCNTs) [10], Rare-Earth Doped Nanoparticles [10]
Protein Carriers Enhance fluorescence brightness and biocompatibility Human Serum Albumin (HSA) [11], Bovine Serum Albumin (BSA) [16]
Encapsulation Matrices Improve aqueous solubility and stability PEG-phospholipids [17], F127 copolymer [17]
Targeting Moieties Enable molecular-specific imaging Antibodies [12], Peptides, Folic Acid [18]
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Workflow and Signaling Pathways

The following diagram illustrates the logical workflow for selecting appropriate NIR-II spectral regions and fluorophores based on specific research applications:

G cluster_app Application Requirements cluster_spectral Spectral Region Selection cluster_probe Probe Selection Start Define Research Objective A1 Deep Tissue Penetration Start->A1 A2 High Spatial Resolution Start->A2 A3 Maximum Contrast Start->A3 A4 Clinical Translation Start->A4 S2 NIR-IIb (1500-1700 nm) Highest Contrast A1->S2  Prioritize S3 NIR-IIx (1400-1500 nm) Water Absorption Enhanced A2->S3  Prioritize A3->S2  Prioritize S4 Off-Peak Detection Leverage NIR-I Dyes A4->S4  Prioritize S1 NIR-IIa (1300-1400 nm) Balanced Performance P1 Organic Small Molecules (TPA-TQT, CH1055) S1->P1 P2 Nanoparticles (QDs, SWCNTs) S1->P2 S2->P1 S2->P2 S3->P2 P4 AIEgens (Aggregation-Induced Emission) S3->P4 P3 Clinical Dyes with NIR-II Tails (ICG, IRDye800CW) S4->P3 End Optimized NIR-II Imaging P1->End P2->End P3->End P4->End

NIR-II Experimental Design Workflow

The precise definition of NIR-II spectral regions and their appropriate application is fundamental to advancing deep-tissue fluorescence imaging research. The standardized subdivisions of the NIR-II window (NIR-IIa, NIR-IIb) provide a framework for selecting optimal imaging parameters based on specific research needs, while newly proposed regions (NIR-IIx, NIR-IIc, NIR-III) suggest future directions for technological development. The experimental protocols outlined herein enable researchers to implement both cutting-edge approaches using specialized NIR-II fluorophores and accessible methods utilizing clinically available dyes with NIR-II emission tails. As the field continues to evolve, the refined understanding of light-tissue interactions across the extended near-infrared spectrum will further enhance the capabilities of in vivo imaging for basic research and drug development applications.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) represents a significant advancement over traditional NIR-I (700–900 nm) imaging for deep-tissue biological investigations. The core advantage of NIR-II bioimaging stems from fundamentally superior light-tissue interactions in this spectral region, which enable researchers to overcome the pervasive challenges of light scattering and absorption in biological materials [1] [19]. When propagating through living tissue, NIR-II photons experience diminished absorption by biomolecules such as hemoglobin and water, reduced tissue autofluorescence, and suppressed photon scattering compared to both visible light and NIR-I radiation [1] [20]. These physical phenomena collectively enable NIR-II fluorescence imaging to achieve deeper tissue penetration (typically reported in the range of 5-20 mm) and a significantly higher signal-to-background ratio (SBR), providing unprecedented clarity and resolution for non-invasive in vivo studies [1] [19]. This application note details the quantitative metrics validating NIR-II superiority and provides standardized protocols for its application in deep-tissue imaging research.

Quantitative Performance Comparison: NIR-II vs. NIR-I

The performance advantages of NIR-II imaging can be quantified across several key metrics. The following tables consolidate experimental data from recent studies, providing researchers with benchmark values for experimental planning and validation.

Table 1: Comparative Optical Properties of NIR-I and NIR-II Windows

Performance Metric NIR-I Window (700-900 nm) NIR-II Window (1000-1700 nm) Experimental Basis
Tissue Penetration Depth < 1 mm [19] 5–20 mm [1] Measured in tissue phantoms and in vivo murine models
Photon Scattering High Significantly reduced (scales as λ-α, α=0.2-4) [11] Turbid medium measurements
Tissue Autofluorescence Significant Minimal/negligible [20] Ex vivo tissue spectroscopy
Spatial Resolution ~10-30 μm at superficial depths Sub-millimeter at >3 mm depth [1] Resolution target imaging through tissue
Signal-to-Background Ratio (SBR) Moderate (e.g., ~2-4 for tumors) [11] High (e.g., 2-4 fold improvement over NIR-I) [11] In vivo tumor imaging with targeted probes

Table 2: Performance of Representative NIR-II Fluorophores

Fluorophore Type Emission Range (nm) Quantum Yield (%) Clearance Pathway Key Applications
Organic D-A-D Dyes (e.g., CH1055) 900-1600 [11] 0.03-0.53 [11] >90% Renal [11] Vascular imaging, tumor targeting
Lanthanide Nanoparticles 1000-1700 [1] Varies by composition MPS/Liver [20] Multiplexed imaging
Quantum Dots (PbS) >1500 [1] High (varies) MPS/Liver [20] Deep-tissue ovary imaging
Repurposed NIR-I Dyes (ICG tail) 1000-1500 [11] N/A (tail emission) Hepatobiliary [20] Clinical translation studies

Experimental Protocols for NIR-II Imaging

Protocol: NIR-II In Vivo Vascular Imaging with Organic Fluorophores

This protocol describes methodology for high-resolution visualization of murine cerebral vasculature using the organic NIR-II fluorophore CH1055-PEG [11].

  • Primary Objective: To achieve non-invasive, high-SBR imaging of deep vasculature.
  • Principle: The D-A-D structured CH1055-PEG fluorophore, upon excitation, emits in the NIR-II window, minimizing light scattering and autofluorescence for superior image clarity.

Materials & Reagents

  • Fluorophore: CH1055-PEG solution (0.1 mM in saline)
  • Animal Model: Nude mouse (25-30 g)
  • Imaging System: NIR-II fluorescence imaging setup with:
    • 808 nm or 980 nm laser source
    • InGaAs CCD camera (sensitive to 1000-1700 nm)
    • 1000 nm long-pass emission filter
  • Anesthesia: Isoflurane vaporizer system
  • Injection Supplies: 29G insulin syringes
  • Software: Image analysis software (e.g., ImageJ with NIR-II plugins)

Procedure

  • Animal Preparation: Anesthetize the mouse using 2% isoflurane. Secure the animal in a stereotaxic frame or supine position on a heating pad (37°C) to maintain body temperature.
  • Dose Administration: Intravenously inject CH1055-PEG via the tail vein at a dose of 2-4 mg/kg (approximately 100-200 µL per mouse).
  • Image Acquisition:
    • Position the animal under the NIR-II imaging system.
    • Set the laser excitation power to 100 mW/cm² at 808 nm.
    • Acquire time-series images at 100-ms exposure times for dynamic angiography.
    • For high-resolution static images, acquire a single frame with 500-ms exposure 5-10 minutes post-injection.
    • Maintain the acquisition settings constant for all comparative studies.
  • Data Analysis:
    • Calculate SBR by selecting a region of interest (ROI) over a vessel and a nearby background tissue region. SBR = (Mean SignalVessel - Mean SignalBackground) / Mean SignalBackground.
    • Measure the full-width at half-maximum (FWHM) of intensity line profiles across vessels to quantify spatial resolution.

Troubleshooting Notes

  • High Background: Ensure the 1000 nm long-pass emission filter is correctly seated to block any excitation light or NIR-I autofluorescence [21].
  • Low Signal: Confirm fluorophore concentration and integrity. Check laser power and camera sensitivity settings.
  • Motion Artifacts: Ensure proper anesthetic depth and use of a stereotaxic frame for brain imaging.

Protocol: Time-Gated Imaging with Long-Lived NIR-II Probes

This protocol leverages the long fluorescence lifetime of certain probes (e.g., lanthanide-doped nanoparticles, ruthenium complexes) to suppress short-lived background via time-gated detection [22].

  • Primary Objective: To isolate the specific NIR-II signal from a short-lived autofluorescence and scattering background.
  • Principle: A pulsed laser excites the sample, and detection is delayed (time-gated) until the instantaneous scattering and short-lived autofluorescence (Ï„ < 10 ns) have decayed, allowing selective detection of the long-lived probe emission (Ï„ > 100 ns).

Materials & Reagents

  • Probe: Long-lifetime NIR-II probe (e.g., Er³+-doped nanoparticle suspension)
  • Imaging System: Time-gated NIR-II fluorescence microscope or imager with:
    • Pulsed laser (e.g., 980 nm, pulse width < 10 ns)
    • InGaAs camera with external trigger/gating capability
  • Sample: Cell spheroid or thin tissue section labeled with the probe.

Procedure

  • System Calibration:
    • Measure the fluorescence lifetime of your probe to determine optimal gate delay.
    • Set the laser to pulse mode with a repetition rate of 100 kHz.
  • Image Acquisition:
    • Set the camera gate delay (td) to 2-3 times the lifetime of the short-lived background (typically 20-50 ns).
    • Set the gate width (tw) to 2-3 times the lifetime of your long-lived probe.
    • Acquire the time-gated image (ITG).
    • For differential imaging, acquire a second image (IBG) with the gate set immediately after the laser pulse to capture primarily background.
  • Data Processing:
    • Generate a background-subtracted image: IFinal = ITG - k·IBG, where k is a normalization factor.
    • This differential image manipulation highly limits background and instrumental noise, resulting in more specific/sensitive detection and allowing for greater imaging depth in highly scattering media [22].

Visualization of NIR-II Advantages

The following diagram illustrates the fundamental principles that give NIR-II imaging its performance advantage, focusing on photon-tissue interactions.

NIRvsNIRII Photon-Tissue Interaction Pathways: NIR-I vs NIR-II LightSource Excitation Light Source NIR_I NIR-I Photon (700-900 nm) LightSource->NIR_I NIR_II NIR-II Photon (1000-1700 nm) LightSource->NIR_II Scattering_I High Scattering NIR_I->Scattering_I ReducedScattering_II Reduced Photon Scattering NIR_II->ReducedScattering_II ShallowPen_I Limited Penetration Depth (<1 mm) Scattering_I->ShallowPen_I Autofluor_I Significant Tissue Autofluorescence Scattering_I->Autofluor_I LowSBR_I Low Signal-to-Background Ratio (SBR) Autofluor_I->LowSBR_I DeepPen_II Deep Tissue Penetration (5-20 mm) ReducedScattering_II->DeepPen_II LowAutofluor_II Minimal Tissue Autofluorescence ReducedScattering_II->LowAutofluor_II HighSBR_II High Signal-to-Background Ratio (SBR) LowAutofluor_II->HighSBR_II

The Scientist's Toolkit: Essential Research Reagent Solutions

Successful NIR-II imaging requires a suite of specialized reagents and materials. The following table details key components for building a robust NIR-II imaging pipeline.

Table 3: Essential Reagents and Materials for NIR-II Bioimaging Research

Item Category Specific Examples Function & Application Notes
NIR-II Fluorophores CH1055-PEG, IR-FGP, FD-108 [11] Organic small molecules for high-resolution vascular imaging and rapid renal clearance. Optimal for acute studies.
Clinical NIR-I/NIR-II Dyes Indocyanine Green (ICG), IRDye800CW [11] Leverage NIR-II "tail" emission (1000-1500 nm) for accelerated clinical translation studies.
Inorganic Nanoparticles Rare-earth doped nanoparticles (RENPs), PbS Quantum Dots [1] High brightness for multiplexed imaging and sensing; note potential long-term retention [20].
Surface Coating Agents DSPE-PEG, PS-PEG, Cyclodextrin [20] Improve biocompatibility, aqueous solubility, and circulation half-life of nanoprobes.
Mounting/Imaging Media FluoroBrite DMEM, OptiClear [21] Specially formulated media with low autofluorescence for live-cell and ex vivo imaging.
Emission Filters 1000 nm, 1300 nm, 1500 nm long-pass filters Critical for blocking excitation light and isolating the NIR-II signal from NIR-I background.
Lhc-165LHC-165LHC-165 is a potent Toll-like receptor 7 (TLR7) agonist for research into intratumoral immunotherapy. For Research Use Only. Not for human or veterinary diagnostic or therapeutic use.
MK-3903MK-3903, CAS:1219737-12-8, MF:C27H19ClN2O3, MW:454.91Chemical Reagent

The transition from NIR-I to NIR-II fluorescence imaging provides a paradigm shift in deep-tissue optical bioimaging, offering quantifiable and significant improvements in both penetration depth and signal-to-background ratio. The experimental protocols and performance metrics outlined in this application note provide a foundational framework for researchers to implement and validate this powerful technology in their investigative workflows. As the palette of biocompatible NIR-II contrast agents continues to expand, incorporating advanced features such as target-specificity and activability, the application of NIR-II imaging is poised to unlock new frontiers in non-invasive biological observation and preclinical drug development.

The emergence of fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) represents a paradigm shift in biomedical optical imaging, addressing fundamental limitations of traditional visible and NIR-I (700–900 nm) techniques. The historical development of this field stems from the recognition that light propagation in biological tissues is governed by wavelength-dependent scattering and absorption phenomena. Compared to the NIR-I window, the NIR-II window offers significantly reduced photon scattering, diminished tissue autofluorescence, and lower light absorption by endogenous chromophores, enabling unprecedented capabilities for deep-tissue in vivo imaging with high spatial resolution and superior signal-to-background ratios (SBR) [4] [1] [23].

The pioneering work in this field dates to 2003 when Professor Frangioni and Professor Bawendi's team first developed near-infrared quantum dots and demonstrated optimized vascular imaging using wavelengths beyond 1000 nm, hinting at the potential of this optical window [23]. However, the critical breakthrough came in 2009 when Welsher et al. utilized surface-functionalized single-walled carbon nanotubes (SWCNTs) as NIR-II fluorescent agents, achieving high-resolution imaging of tumor vasculature through thick skin layers with minimal background interference [23]. This seminal study established the foundation for NIR-II bioimaging and ignited rapid development of both imaging instrumentation and contrast agents spanning inorganic nanomaterials, organic fluorophores, and molecular complexes [4] [1] [23].

Pioneering Contrast Agents and Their Evolution

Inorganic Nanomaterials

Carbon Nanotubes

The initial demonstration of NIR-II imaging employed polyethylene glycol (PEG)-modified SWCNTs, which exhibited inherent photoluminescence in the NIR-II region alongside excellent biocompatibility. This pioneering approach revealed the practical advantage of NIR-II imaging: deep tissue penetration with low autofluorescence background, enabling clear visualization of anatomical features previously challenging for optical techniques [23].

Quantum Dots (QDs)

Following carbon nanotubes, quantum dots emerged as versatile NIR-II probes with tunable emission properties. Various QD compositions have been developed for biomedical imaging applications:

  • Lead Sulfide (PbS) QDs: PEGylated core-shell PbS/CdS QDs with emissions tunable from 1100 to 1700 nm have enabled high-contrast in vivo imaging, even in challenging spectral regions near water absorption peaks (~1450 nm and ~1930 nm) [24].
  • Silver Chalcogenide QDs: Agâ‚‚S and Agâ‚‚Se QDs offer emissions between 950–1400 nm and demonstrate favorable biocompatibility and clearance profiles [25] [23].
  • Other QD Systems: Cadmium-based (II-VI group), indium arsenide (III-V group), and carbon/silicon (IV group) QDs have also been explored, though clinical translation of heavy metal-containing QDs requires careful toxicological assessment [25].

The table below summarizes the properties and applications of major inorganic NIR-II probes:

Table 1: Evolution of Major Inorganic NIR-II Fluorescence Probes

Nanomaterial Emission Range (nm) Key Advantages Representative Applications Key References
Single-Walled Carbon Nanotubes 1000–1400+ First demonstrated NIR-II in vivo imaging; high photostability Vasculature imaging, tumor detection [23]
PbS/CdS QDs 1100–1700 Bright, tunable emission; core-shell structure improves stability Deep-tissue imaging in multiple sub-windows (NIR-IIb, NIR-IIc) [24]
Ag₂S QDs 1000–1400 Improved biocompatibility; lower toxicity Early-stage tumor diagnosis, blood clearance studies [25] [23]
Rare-Earth Doped Nanoparticles 980–1600 Narrow emission bands; suitable for multiplexed imaging Lymph node mapping, vascular imaging [1]

Organic Fluorophores and Small Molecules

While inorganic probes paved the way, organic fluorophores offer potential advantages in biodegradability, pharmacokinetics, and synthetic tunability. Key developments include:

  • Indocyanine Green (ICG): Although primarily a NIR-I dye, ICG exhibits tail emission into the NIR-II window and has been used for clinical fluorescence angiography and NIR-II microsurgery [26] [27]. However, it suffers from low NIR-II quantum yield (0.1–0.3%) and concentration-dependent aggregation [27].
  • Donor-Acceptor-Donor (D-A-D) Dyes: Molecular engineering of structures with strong electron donors and acceptors connected via Ï€-conjugated bridges has produced fluorophores with emissions extending beyond 1000 nm, offering high extinction coefficients and improved quantum yields [27] [28].
  • Aggregation-Induced Emission (AIE) Nanoparticles: AIE luminogens (AIEgens) exploit restricted intramolecular motion in aggregate state to enhance emission, overcoming aggregation-caused quenching. They have been utilized in hybrid NIR-II/visible navigation systems for surgery [4] [29].

Experimental Protocols: From Probe Synthesis to Imaging Validation

Protocol 1: Synthesis and Biocompatibility Modification of PbS/CdS Core/Shell QDs

This protocol outlines the synthesis of bright, water-soluble PbS/CdS QDs for deep NIR-II imaging [24].

Materials:

  • Lead oxide (PbO)
  • Oleic acid (OA)
  • 1-Octadecene (ODE)
  • Bis(trimethylsilyl)sulfide ((TMS)â‚‚S)
  • Cadmium oxide (CdO)
  • Polyethylene glycol (PEG)-functionalized ligands

Procedure:

  • PbS Core Synthesis: Heat a mixture of PbO, OA, and ODE to 150°C under argon flow until complete dissolution. Cool to 120°C and swiftly inject (TMS)â‚‚S dissolved in ODE. Allow reaction to proceed for 2–5 minutes before cooling to room temperature. Purify the crude solution by precipitation with acetone.
  • CdS Shell Growth: Prepare a cadmium-oleate precursor by heating CdO in OA and ODE. Redisperse the purified PbS cores in ODE and heat to 100°C. Slowly inject the cadmium-oleate precursor, then raise the temperature to 140°C for shell growth over 1–2 hours.
  • Aqueous Phase Transfer: Ligand exchange is performed by reacting the hydrophobic PbS/CdS QDs with PEG-containing mercaptoaniline ligands in a mixture of chloroform and dimethylformamide. Transfer to aqueous solution and purify via dialysis or centrifugation.
  • Characterization: Perform transmission electron microscopy (TEM) for size and morphology analysis. Use UV-Vis-NIR spectroscopy and fluorescence spectrometry to determine absorption and emission profiles. Measure photoluminescence quantum yield using an integrating sphere.

Protocol 2: Back Table NIR-II Fluorescence Imaging for Surgical Guidance

This protocol describes intraoperative assessment of tissue specimens using clinical NIR-II imaging systems like the LightIR platform [26].

Materials:

  • LightIR imaging system or equivalent clinical NIR-II imager
  • NIR-II fluorescent probe (e.g., ICG, IR-1048, or targeted nanoprobes)
  • Animal model or freshly excised human tissue specimen
  • Anesthesia equipment (for in vivo studies)
  • Phosphate buffered saline (PBS)

Procedure:

  • Probe Administration: For in vivo imaging, systemically administer the NIR-II fluorophore via intravenous injection at a predetermined dosage (e.g., ICG at 200 µM/kg). Allow appropriate circulation time for target accumulation.
  • Specimen Preparation: For back table imaging, surgically excise the target tissue and rinse gently with PBS to remove surface blood. Place the specimen on the imaging stage under ambient light conditions.
  • Imaging Setup: Position the LightIR system approximately 20–30 cm above the specimen. The system operates in pulse-mode under ambient lighting without requiring a blackout enclosure.
  • Image Acquisition: Use real-time background subtraction algorithms to enhance contrast. Acquire fluorescence images using appropriate long-pass filters (e.g., LP1000, LP1250, LP1500) corresponding to the fluorophore's emission. Adjust exposure time (may require longer exposures than enclosed systems) and laser power to optimize signal-to-noise ratio.
  • Image Analysis: Quantify fluorescence intensity, signal-to-background ratio, and spatial resolution. Compare tumor-to-normal tissue contrast to guide surgical decision-making regarding margin status.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 2: Key Research Reagent Solutions for NIR-II Imaging Studies

Item Function/Application Examples/Specifications
NIR-II Fluorophores Provides contrast for deep-tissue imaging ICG (clinical), IR-1048, PbS/CdS QDs, Agâ‚‚S QDs, D-A-D dyes, AIEgens
Targeting Ligands Enables specific accumulation in diseased tissues Antibodies, peptides (e.g., RGD), carbohydrates, small molecules
Surface Modification Agents Improves biocompatibility and circulation PEG derivatives, phospholipids, multidentate polymers, proteins (e.g., BSA)
Imaging Systems Detection of NIR-II fluorescence IR VIVO (preclinical), LightIR (clinical), InGaAs detectors (640×512 pixels)
Surgical Navigation Tools Intraoperative guidance Hybrid NIR-II/visible imaging systems, endoscopy-compatible systems
Long-Pass Filters Spectral separation of excitation and emission LP1000, LP1250, LP1500 (nm cut-on wavelengths)
ML-102-(5-Fluoropentyl)-2-methylmalonic Acid|ML-10 PrecursorResearch-grade 2-(5-Fluoropentyl)-2-methylmalonic acid, the tosylate precursor for synthesizing the PET apoptosis tracer 18F-ML-10. For Research Use Only. Not for human or veterinary diagnostic or therapeutic use.
NH-bis(PEG3-azide)NH-bis(PEG3-azide), CAS:1258939-39-7, MF:C16H33N7O6, MW:419.48 g/molChemical Reagent

Visualization of Experimental Workflows and Biological Pathways

Workflow for NIR-II Probe Development and Application

The following diagram illustrates the logical workflow from probe design to biomedical application in NIR-II research.

workflow Start Probe Design and Synthesis Characterization Physicochemical Characterization Start->Characterization Targeting Surface Modification and Targeting Characterization->Targeting Characterization_sub Emission Wavelength Quantum Yield Biocompatibility Characterization->Characterization_sub Validation In Vitro and In Vivo Validation Targeting->Validation Application Biomedical Application Validation->Application Validation_sub Biodistribution Toxicity Assessment Imaging Performance Validation->Validation_sub Application_sub Tumor Resection Vascular Imaging Therapeutic Monitoring Application->Application_sub

Diagram 1: NIR-II Probe Development Workflow

Mechanism of NIR-II Fluorescence Imaging Advantage

This diagram contrasts the photon propagation and image formation mechanisms in NIR-I versus NIR-II windows.

mechanism cluster_NIRI NIR-I Imaging (700-900 nm) cluster_NIRII NIR-II Imaging (1000-1700 nm) NIRI_Photon Higher Scattering Shorter Mean Free Path NIRI_Auto Significant Tissue Autofluorescence NIRI_Photon->NIRI_Auto NIRI_Result Blurred Image Lower Contrast Limited Penetration NIRI_Auto->NIRI_Result NIRII_Photon Reduced Scattering Longer Mean Free Path NIRII_Auto Minimal Tissue Autofluorescence NIRII_Photon->NIRII_Auto NIRII_Result Sharper Image Higher SBR Deeper Penetration NIRII_Auto->NIRII_Result Input Excitation Light Input->NIRI_Photon Input->NIRII_Photon

Diagram 2: NIR-I vs. NIR-II Imaging Mechanisms

Current Challenges and Future Perspectives in Clinical Translation

Despite remarkable progress, the clinical translation of NIR-II imaging faces several challenges. Existing NIR-II fluorescent probes still confront issues of insufficient fluorescence brightness, limited wavelength tuning, and suboptimal biocompatibility [4]. The complex synthesis processes, lack of target specificity, and potential long-term metabolic safety risks further complicate clinical adoption [4] [23]. For inorganic probes, particularly those containing heavy metals, comprehensive toxicological profiles and clearance pathways require further investigation [25] [23].

Future developments will likely focus on multiscale molecular design strategies to enhance probe performance [4]. The synergistic combination of NIR-II fluorescence imaging with other modalities like photoacoustic imaging, MRI, and CT will provide complementary information for more precise diagnostics [27] [1]. Furthermore, the integration of NIR-II imaging with therapeutic interventions—creating true theranostic platforms—represents a promising frontier. As probe chemistry and imaging hardware continue to co-evolve, the vision of NIR-II fluorescence imaging becoming a standard tool for precision surgery and deep-tissue diagnosis moves closer to clinical reality [26] [29] [23].

Toolkit for Innovation: Fluorophores, Imaging Systems, and Breakthrough Applications

Near-infrared-II (NIR-II, 1000–1700 nm) fluorescence imaging has emerged as a transformative biomedical modality that significantly overcomes the limitations of conventional imaging in the visible (400–700 nm) and first near-infrared (NIR-I, 700–900 nm) windows [30] [31]. Imaging in the NIR-II window benefits from markedly reduced photon scattering, minimal tissue autofluorescence, and lower light absorption by endogenous chromophores such as hemoglobin and water [30] [32]. This synergistic effect enables deeper tissue penetration (up to several centimeters), improved spatial resolution (micron-level), and significantly enhanced signal-to-background ratios (SBR) compared to NIR-I imaging [33] [32]. These advantages make NIR-II imaging particularly powerful for a wide range of preclinical applications, including deep-tissue tumor imaging, cerebrovascular angiography, and image-guided surgery [34] [35]. The development of high-performance fluorescent probes is crucial to fully exploiting the potential of this imaging modality. This review comprehensively outlines the current landscape of NIR-II fluorophores, categorizing them into organic molecules, inorganic nanomaterials, and activatable probes, while providing detailed experimental protocols for their application in deep-tissue imaging research.

Categories of NIR-II Fluorophores

Organic Small-Molecule Fluorophores

Organic small-molecule fluorophores represent one of the most promising classes of NIR-II contrast agents due to their well-defined chemical structures, excellent biocompatibility, favorable pharmacokinetics, and potential for clinical translation [30] [7] [32]. Most are designed with specific molecular architectures that promote intramolecular charge transfer (ICT) to reduce the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), thereby enabling redshifted absorption and emission [30] [36].

  • Donor-Acceptor-Donor (D-A-D) Frameworks: These fluorophores feature a strong electron-withdrawing core (Acceptor) symmetrically linked to electron-donating units (Donors) via Ï€-conjugated bridges [32]. The benzobisthiadiazole (BBTD) unit is a prototypical acceptor, known for its strong electron-withdrawing character and narrow bandgap [32]. Molecular engineering strategies, such as enhancing donor strength, reducing the D-A distance, and incorporating sterically bulky shielding units (e.g., polyethylene glycol (PEG) chains), are employed to redshift the emission, suppress non-radiative decay, and improve the fluorescence quantum yield (QY) [30] [36]. For instance, engineering a D-A-D molecule (2FT-oCB) with a reduced D-A distance and twisted structure resulted in an emission peak at 1215 nm and an ultralong molecular packing distance exceeding 8 Ã…, effectively preventing Ï€-Ï€ stacking and aggregation-caused quenching (ACQ) [36].

  • Cyanine Derivatives: Cyanine dyes feature a polymethine chain connecting two nitrogen-containing heterocyclic units [32]. While classic dyes like indocyanine green (ICG) have tail emissions in the NIR-II, their QYs are typically low [37]. A breakthrough bioengineering strategy involves complexing cyanine dyes (e.g., IR-783) with recombinant human serum albumin (HSA) domains, particularly domain III (DIII). This "hitchhiking" strategy stabilizes the dye in a hydrophobic pocket, restricting molecular motion and shielding it from aqueous quenching, which dramatically enhances fluorescence brightness and photostability [37].

  • Aggregation-Induced Emission (AIE) Luminogens: To overcome the common ACQ effect, AIE-active NIR-II fluorophores are designed. These molecules are non-emissive in solution but become highly fluorescent in their aggregated state, as the restriction of intramolecular motion (vibrations and rotations) blocks non-radiative decay pathways [30] [36]. This property is highly beneficial for formulating nanoparticle probes that accumulate at disease sites.

Table 1: Key Characteristics of Organic Small-Molecule NIR-II Fluorophores

Structural Class Representative Examples Emission Range (nm) Key Advantages Primary Challenges
D-A-D BBTD-based dyes (e.g., 2FT-oCB) [32] [36] 1000–1300+ Tunable structure, good biocompatibility, relatively high QY Can be hydrophobic, prone to ACQ without design mitigation
Cyanine ICG, IR-783, IR-783@DIII complex [32] [37] 900–1100+ Some are clinically approved (ICG), modifiable Low QY in free state, poor photostability
AIE Luminogens TPE-based D-A-D molecules [30] 1000–1300 High brightness in aggregate/ solid state, resists ACQ Synthetic complexity, potential for large batch-to-batch variance
BODIPY/Xanthene Modified BODIPY dyes [32] 900–1100 High molar extinction coefficients, good photostability Difficulty in achieving emission >1100 nm
Chitinase-IN-1Chitinase-IN-1, MF:C18H16N4O2S, MW:352.4 g/molChemical ReagentBench Chemicals
1-Stearo-3-linolein1-Stearo-3-linolein, MF:C39H72O5, MW:621.0 g/molChemical ReagentBench Chemicals

Inorganic and Hybrid Nanomaterial Fluorophores

Inorganic nanomaterials offer high photostability, strong fluorescence intensity, and tunable optical properties, making them valuable for demanding imaging applications [34] [35].

  • Quantum Dots (QDs): Semiconductor QDs like Agâ‚‚S, PbS, and Cd-based QDs exhibit size-tunable NIR-II emission, high QYs, and broad absorption profiles [34] [35]. Agâ‚‚S QDs are particularly notable for their low toxicity and emission tunability from 687 to 1294 nm [35]. However, the long-term toxicity and potential bioaccumulation of heavy metals remain significant concerns for clinical translation [30] [35].

  • Rare-Earth-Doped Nanoparticles (RENPs): RENPs are typically composed of an inorganic host matrix doped with lanthanide ions (e.g., Yb³⁺, Er³⁺, Nd³⁺). They exhibit unique optical properties, including large Stokes shifts, narrow emission bands, long luminescence lifetimes (micro- to milliseconds), and minimal photobleaching, which are ideal for multiplexed and lifetime-based imaging [34] [1] [35]. Core-shell-shell structures can be designed to enhance emission intensity at specific wavelengths, such as 1525 nm [35].

  • Single-Walled Carbon Nanotubes (SWCNTs): Semiconducting SWCNTs were among the first materials used for NIR-II bioimaging. They display photostable fluorescence across a broad range of the NIR-II spectrum (1000–1600 nm) [31] [35]. Their surface can be functionalized with phospholipid-PEG to improve water solubility and biocompatibility, enabling applications in vascular imaging [35].

  • Metal Nanoclusters (MNCs): Gold nanoclusters (Au NCs) consist of a few to hundreds of gold atoms. They are characterized by good biocompatibility, renal clearance potential, and tunable emission extending into the NIR-II window [34] [35]. Their surface chemistry allows for functionalization with therapeutic agents, enabling theranostic applications [35].

Table 2: Key Characteristics of Inorganic NIR-II Fluorophores

Material Class Representative Examples Emission Range (nm) Key Advantages Primary Challenges
Quantum Dots (QDs) Ag₂S QDs, PbS QDs [34] [35] 900–1300+ High QY, size-tunable emission, photostable Potential heavy metal toxicity, long-term retention
Rare-Earth Nanoparticles (RENPs) NaYF₄:Yb/Er/Nd @ NaYF₄ [34] [1] 1000–1600 Narrow bands, long lifetimes, low background Lower QY than QDs, complex synthesis
Carbon Nanotubes PEGylated SWCNTs [31] [35] 1000–1600 Ultra-broad emission, high photostability High aspect ratio, potential fiber-like toxicity
Metal Nanoclusters Au₂₅, Au/Cu alloy clusters [34] [35] 900–1200 Good biocompatibility, renal clearable, tunable Relatively low QY, complex synthesis

Activatable Molecular Probes

A significant limitation of many NIR-II fluorophores is their "always-on" signal, which can lead to high background noise and reduced specificity [30] [7]. Activatable (or "smart") probes address this issue by remaining in a silent ("off") state until activated by a specific disease-associated biomarker, resulting in a fluorescence turn-on ("on") response at the target site [30] [7].

These probes are engineered to exploit pathological stimuli in the tumor microenvironment (TME) or other diseased tissues. The primary activation mechanisms include:

  • Enzymatic Activity: Cleavage of a specific peptide sequence by overexpressed enzymes (e.g., matrix metalloproteinases, cathepsins) releases fluorescence quenching or alters the probe's electronic structure [30].
  • pH Variation: The acidic TME (pH 6.5–6.9) can trigger the protonation of acid-labile bonds on the probe, leading to disassembly or structural changes that activate fluorescence [30].
  • Redox Reactions: The high concentration of reactive oxygen species (ROS) or glutathione (GSH) in diseased cells can cleave specific chemical bonds (e.g., thioketal, disulfide bonds), activating the probe [30].
  • Other Stimuli: Probes responsive to hypoxia, viscosity, and specific ions (e.g., Zn²⁺) have also been developed, allowing for the mapping of diverse physiological parameters [30].

Experimental Protocols for NIR-II Bioimaging

Protocol 1: NIR-II Fluorescence Imaging of Tumor-Bearing Mice

Purpose: To non-invasively visualize and quantify tumor location, morphology, and biomarker activity in live animal models.

Materials:

  • NIR-II Fluorophore: e.g., D-A-D small molecule (1 mg/mL in saline with <5% DMSO), or inorganic nanoprobe (e.g., Agâ‚‚S QDs, 100 µM in PBS).
  • Animal Model: Immunocompetent or immunodeficient mice bearing subcutaneous or orthotopic tumors.
  • Imaging System: NIR-II fluorescence imaging system equipped with a 808 nm or 980 nm laser diode, appropriate long-pass filters (1000 nm, 1500 nm LP), and an InGaAs camera.
  • Anesthesia System: Isoflurane vaporizer and induction chamber.
  • Software: For image acquisition and analysis (e.g., MATLAB, ImageJ).

Procedure:

  • Animal Preparation: Anesthetize the mouse using 2–3% isoflurane in oxygen. Place the mouse in a prone or lateral position on the heated imaging stage, maintaining anesthesia at 1.5–2% isoflurane.
  • Baseline Imaging: Acquire a pre-injection image of the mouse using the same parameters planned for post-injection imaging (laser power: 50–100 mW/cm²; exposure time: 100–500 ms; filter: 1500 nm LP for long-wavelength imaging).
  • Probe Administration: Intravenously inject the NIR-II fluorophore via the tail vein at a dose of 100–200 µL (e.g., 2–5 mg/kg for organic dyes, 1–10 nM for nanoparticles).
  • Time-Point Imaging: Acquire images at multiple time points post-injection (e.g., 1, 6, 12, 24, 48 hours) using consistent imaging parameters. For kinetics studies, acquire dynamic images immediately after injection.
  • Image Analysis:
    • Region of Interest (ROI): Draw ROIs over the tumor and contralateral background tissue.
    • Signal-to-Background Ratio (SBR): Calculate as SBR = Mean Fluorescence Intensity (Tumor) / Mean Fluorescence Intensity (Background).
    • Pharmacokinetics: Plot fluorescence intensity in the tumor ROI over time to determine the optimal imaging time window.
  • Ex Vivo Validation: At the endpoint, euthanize the mouse, collect the tumor and major organs (liver, spleen, kidney, etc.), and image them ex vivo to confirm probe distribution and assess biodistribution.

Protocol 2: Evaluating an Enzyme-Activatable NIR-II Probe

Purpose: To validate the specific activation of a smart probe by its target enzyme, both in vitro and in vivo.

Materials:

  • Activatable Probe: e.g., a probe with a quencher linked via an enzyme-cleavable peptide sequence.
  • Target Enzyme: Recombinant enzyme (e.g., MMP-2, 0.1–1 µg/µL).
  • Control: Inactive enzyme or specific enzyme inhibitor.
  • Buffer: Suitable reaction buffer (e.g., PBS, pH 7.4).
  • NIR-II Spectrofluorometer or Plate Reader.

Procedure:

  • Part A: In Vitro Activation Assay
    • Prepare three solutions in a 96-well plate or quartz cuvette:
      • Experimental: 100 µL of probe (1 µM) + 10 µL of target enzyme.
      • Control 1: 100 µL of probe (1 µM) + 10 µL of buffer.
      • Control 2: 100 µL of probe (1 µM) + 10 µL of heat-inactivated enzyme.
    • Incubate the plate/cuvettes at 37°C.
    • Measure the NIR-II fluorescence spectrum (e.g., 1000–1700 nm) at regular intervals (e.g., 0, 15, 30, 60, 120 min) using an excitation wavelength matching the probe's absorption peak.
    • Plot fluorescence intensity at the emission maximum versus time. A significant increase in the experimental group only confirms enzyme-specific activation.
  • Part B: In Vivo Imaging
    • Follow Protocol 1 to prepare and image tumor-bearing mice.
    • Compare the SBR in mice injected with the activatable probe to control groups injected with an "always-on" version of the probe or after pre-treatment with an enzyme inhibitor.
    • The activatable probe should show a significantly higher tumor SBR due to lower background signal and specific activation at the tumor site.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for NIR-II Fluorescence Imaging

Reagent/Material Function/Description Example Application
Indocyanine Green (ICG) FDA-approved cyanine dye with NIR-II tail emission [32] [37] Clinical translation benchmark, vascular imaging
Recombinant Albumin Domains (e.g., DIII) Engineered protein domains that bind and enhance cyanine dye brightness [37] Creating high-performance, biocompatible fluorophores from clinical dyes
PEGylated Phospholipids Amphiphilic polymers for encapsulating and solubilizing hydrophobic fluorophores or nanomaterials [35] Surface functionalization of SWCNTs, QDs, and organic NPs for improved biocompatibility
BBTD Acceptor Core Strong electron-accepting unit for constructing D-A-D fluorophores [32] [36] Synthesis of small-molecule NIR-II fluorophores with emissions >1000 nm
Agâ‚‚S Quantum Dots Low-toxicity inorganic nanoprobe with tunable NIR-II emission [35] High-resolution deep-tissue and vascular imaging
NaYFâ‚„:Yb/Er/Nd Nanoparticles RENPs with long lifetime and narrow emission bands [34] [1] Multiplexed imaging, lifetime imaging
NIR-II Fluorescence Imager InGaAs camera-based system with NIR lasers and filters [31] Essential equipment for all in vivo and in vitro NIR-II imaging studies
Cycloshizukaol ACycloshizukaol A, CAS:150033-85-5, MF:C32H36O8Chemical Reagent
9-cis-Retinol-d59-cis-Retinol-d5, MF:C20H30O, MW:291.5 g/molChemical Reagent

Visualization of Key Concepts

NIR-II Probe Activation Mechanisms

G Stimuli Pathological Stimuli Probe Inactive Probe ('Off' State) Stimuli->Probe  Triggers Activated Activated Probe ('On' State) Probe->Activated  Molecular  Change Enzyme Enzyme Overexpression Enzyme->Stimuli pH Acidic pH pH->Stimuli Redox Redox Imbalance Redox->Stimuli

Diagram Title: Activation Mechanisms of Smart NIR-II Probes

Workflow for In Vivo NIR-II Imaging

G Start Animal Model Preparation (Tumor-bearing mouse) A Anesthetize Animal Start->A B Acquire Baseline NIR-II Image A->B C IV Inject NIR-II Fluorophore B->C D Acquire Time-Point Images (1, 6, 24 h) C->D E Image Analysis (ROI, SBR, Kinetics) D->E F Ex Vivo Validation (Tumor & Organs) E->F

Diagram Title: In Vivo NIR-II Imaging Experiment Workflow

The NIR-II fluorophore landscape is richly diverse, encompassing organic small molecules with high translational potential, robust inorganic nanomaterials for multiplexed imaging, and sophisticated activatable probes for unprecedented specificity. The choice of fluorophore hinges on the specific research question, balancing factors such as emission wavelength, brightness, biocompatibility, and functional design. The experimental protocols outlined provide a foundational roadmap for researchers to conduct high-quality NIR-II bioimaging studies. As molecular engineering and bioengineering strategies continue to advance, the next generation of NIR-II probes will undoubtedly unlock deeper insights into biological processes and accelerate the development of precision diagnostics and therapeutics.

Bioimaging in the second near-infrared window (NIR-II, 950–1700 nm) represents a transformative approach for non-invasive, real-time investigation of biological processes with exceptional spatiotemporal resolution and deep tissue penetration [33]. This imaging modality has demonstrated significant advantages over conventional UV-Vis-NIR and NIR-I windows, primarily due to reduced photon scattering, minimal tissue autofluorescence, and diminished light absorption by tissue chromophores such as hemoglobin and water [38]. These physical properties enable researchers to achieve deeper tissue penetration (5–20 mm) while maintaining high spatial resolution, making NIR-II imaging particularly valuable for preclinical research, drug development, and emerging clinical applications [1] [39].

The evolution of NIR-II imaging instrumentation has progressed along two complementary trajectories: advanced detection systems capable of capturing faint NIR-II signals, and integrated navigation platforms that translate this information into surgical guidance. InGaAs-based cameras have emerged as the gold standard for detecting NIR-II fluorescence, offering superior sensitivity in the 900-1700 nm range compared to silicon-based detectors [40] [39]. Concurrently, augmented reality surgical navigation systems have developed to leverage this deep-tissue imaging capability, providing surgeons with intuitive, real-time visualization of subsurface anatomical structures and pathological features [41]. This combination of sophisticated detection and visualization technologies is rapidly advancing the field of image-guided interventions and precision medicine.

NIR-II Imaging Fundamentals and Physical Principles

Optical Properties in the NIR-II Window

The exceptional performance of NIR-II bioimaging stems from fundamental interactions between light and biological tissues. As photons traverse tissue, they undergo both absorption and scattering events, which collectively determine the penetration depth and image resolution achievable at different wavelengths. In the NIR-II window, scattering is significantly reduced compared to visible and NIR-I wavelengths, leading to clearer images with enhanced spatial resolution [40]. Table 1 quantifies the key advantages of NIR-II imaging relative to other spectral windows.

Table 1: Comparison of Imaging Spectral Windows

Parameter Visible (400-700 nm) NIR-I (700-900 nm) NIR-II (1000-1700 nm)
Penetration Depth Shallow (1-2 mm) Moderate (2-5 mm) Deep (5-20 mm)
Spatial Resolution Limited by scattering Moderate High (∼μm)
Tissue Autofluorescence High Moderate Low
Photon Scattering Significant Moderate Reduced
Absorption by Hemoglobin High Moderate Low

The diminished scattering in the NIR-II window directly translates to superior image clarity. As illustrated in practical experiments, NIR-I wavelengths tend to scatter extensively, resulting in 'fuzzier' images, while NIR-II/SWIR wavelengths yield 'crisper' images with well-defined vascular anatomy [40]. This scattering reduction enables researchers to visualize fine biological structures including capillary networks, tumor vasculature, and neural anatomy with unprecedented detail in thick, biologically relevant tissues [39].

Signal-to-Background Ratio and Contrast Mechanisms

Beyond improved resolution, NIR-II imaging offers enhanced signal-to-background ratio (SBR) due to significantly reduced tissue autofluorescence. Many biological compounds exhibit natural fluorescence in the visible spectrum (collagen, elastin, NADPH), while water absorption peaks around 900-1000nm further complicate NIR-I imaging [39]. The NIR-II window largely avoids these confounding factors, resulting in cleaner signals with higher contrast between target and background tissues.

Contrast in NIR-II imaging can be further enhanced through specialized probes designed to accumulate in specific tissues or respond to pathological conditions. Activatable molecular probes that remain silent until reaching their target environment are particularly valuable for improving diagnostic accuracy by minimizing background signal [7]. The combination of favorable tissue optical properties and advanced contrast agents enables researchers to achieve robust, high-fidelity imaging in complex biological systems.

Instrumentation Platforms: From Detection to Visualization

Detector Technologies for NIR-II Imaging

The choice of detector technology critically influences the performance and applicability of NIR-II imaging systems. Standard silicon-based CCD and sCMOS detectors, while excellent for visible and NIR-I imaging, exhibit rapidly declining quantum efficiency beyond 700nm, typically falling to 20-40% at 900nm [39]. This limitation stems from the fundamental properties of silicon, where longer wavelengths often pass through the photosensitive layer without generating photoelectrons.

Table 2: Performance Comparison of NIR-II Detector Technologies

Detector Type Spectral Range Quantum Efficiency Key Advantages Limitations
Si-CCD (Deep Depleted) 400-900 nm ~50% at 850nm Compatible with visible imaging; reduced etaloning Limited to <1000nm; slower readout
InGaAs Camera 900-1700 nm >70% (900-1700nm) [39] Broad NIR-II coverage; fast frame rates (up to 600fps) [39] Requires cooling; higher cost
Scientific-Grade InGaAs 900-1700 nm ~80-85% (950-1100nm) [42] Deep cooling (-85°C); low dark noise; precision exposure control [40] Specialized infrastructure; higher cost

For imaging beyond 1000nm, Indium Gallium Arsenide (InGaAs) detectors have emerged as the preferred technology. These detectors typically offer quantum efficiency greater than 70% across the entire NIR-II window (900-1700nm), with scientific-grade cameras achieving 80-85% in the 950-1100nm range [42] [39]. This high sensitivity is crucial for detecting the relatively weak signals that emerge from deep tissues after significant attenuation.

A critical consideration for InGaAs cameras is the management of dark current, which can be substantial at room temperature. Advanced scientific InGaAs cameras incorporate maintenance-free thermoelectric cooling systems capable of reaching -85°C, coupled with proprietary cold shield designs and vacuum technology to minimize dark noise [40]. This deep cooling preserves signal-to-noise ratio during long exposures, enabling researchers to capture faint signals from deep tissue structures.

Performance Considerations in Deep-Tissue Imaging

Despite the higher quantum efficiency of InGaAs cameras compared to silicon detectors, an intriguing paradox emerges in deep-tissue imaging scenarios. Under identical imaging conditions using MgGeO3:Yb3+ short-wave infrared persistent luminescent phosphor as a contrast agent, InGaAs cameras surprisingly exhibited "notably inferior performance overall in imaging deep-tissue features" compared to Si CCD cameras when imaging through thick chicken breast tissues (5–20 mm) and mice bodies (10–20 mm) [42]. This counterintuitive finding was attributed to the "pronounced interference of [the] inherently high dark current" in InGaAs detectors, particularly problematic when dealing with faint imaging signals [42].

This performance limitation can be overcome with sufficiently intense SWIR imaging signals, where the InGaAs camera outperforms the Si camera in terms of clarity in tissues up to 10mm thick [42]. This finding highlights the importance of matching detector technology to specific experimental requirements, considering factors such as expected signal intensity, tissue depth, and required frame rate.

Augmented Reality Surgical Navigation Systems

The translation of NIR-II imaging data into surgical guidance has led to the development of augmented reality (AR) surgical navigation systems. These platforms integrate preoperative imaging data with real-time surgical visualization, creating an intuitive interface for navigating complex anatomical relationships. One such system proposed for extra-ventricular drainage (EVD) surgery provides "intuitive guidance for the surgical target point, entry point, and trajectory" through AR devices (tablet or HoloLens glasses) [41].

These AR navigation systems employ "virtual object automatic scanning" to achieve remarkable accuracy of 1 ± 0.1 mm, addressing the stability and precision limitations that have historically hampered surgical AR applications [41]. Furthermore, the integration of deep learning-based U-Net segmentation networks enables automatic identification of pathological features such as hydrocephalus locations, with reported recognition accuracy, sensitivity, and specificity of 99.93%, 93.85%, and 95.73%, respectively [41]. This combination of precise registration and automated segmentation creates a powerful platform for translating NIR-II imaging data into surgical action.

G Start Start: Clinical Need Definition Preop Preoperative Imaging (NIR-II contrast agent administration) Start->Preop Segmentation AI Segmentation (U-Net network) Preop->Segmentation Registration AR Registration (Virtual object automatic scanning) Segmentation->Registration Navigation AR Surgical Navigation (Real-time trajectory guidance) Registration->Navigation Completion Procedure Completion Navigation->Completion

Diagram 1: AR Surgical Navigation Workflow. This flowchart illustrates the integrated process from clinical need definition through procedure completion, highlighting key technological components including AI segmentation and AR registration.

Research Reagent Solutions for NIR-II Imaging

The effectiveness of NIR-II imaging depends critically on the contrast agents employed, with ongoing development focusing on improving brightness, biocompatibility, and target specificity. Table 3 summarizes major categories of NIR-II contrast agents and their characteristics.

Table 3: NIR-II Contrast Agent Classes and Properties

Probe Type Emissions (nm) Key Advantages Limitations Representative Applications
Single-Walled Carbon Nanotubes (SWNTs) 1000-1400+ Good emissivity; can be PEGylated for reduced toxicity [40] Potential long-term retention concerns Vascular imaging [40]
Rare Earth-Doped Nanoparticles Tunable: 950-1100nm (MgGeO3:Yb3+) [42] Low toxicity; narrow emission bands; persistent luminescence [42] [1] Complex synthesis; potential heavy metal content Deep-tissue imaging [42]
Quantum Dots Tunable via size [40] Bright emissions; size-tunable properties Potential heavy metal toxicity; clearance concerns Tumor imaging [40]
Organic Fluorophores 1000-1300+ Well-defined structures; tunable properties; better biocompatibility [7] Generally lower brightness than inorganic options Targeted molecular imaging [7]
Indocyanine Green (ICG) ~800-850nm (extends to NIR-II) [39] FDA-approved; established safety profile Limited to shorter NIR-II wavelengths Clinical perfusion imaging [39]

Recent innovations in contrast agent design have focused on "activatable molecular probes" that address the limitation of "always-on" fluorescence, which leads to background noise and compromised diagnostic accuracy [7]. These smart probes remain quiescent until activated by specific pathological conditions such as abnormal enzyme activity or pH changes, significantly improving target-to-background ratio for precise disease detection.

For multiplexed imaging applications, researchers have developed probes with distinct emission profiles or lifetime characteristics, enabling simultaneous tracking of multiple biological targets. Doped rare-earth nanoparticles are particularly valuable for this application, offering narrow emission bands that can be separated with appropriate optical filters [1]. This multiplexing capability is invaluable for studying complex biological processes involving multiple cell types or molecular targets.

Experimental Protocols and Methodologies

Protocol: In Vivo NIR-II Imaging of Vascular Anatomy

This protocol describes methodology for high-resolution imaging of vascular anatomy in small animal models, adapted from published studies [40] [39].

Materials Required:

  • Scientific-grade InGaAs camera system (e.g., NIRvana or C-RED-2) with 900-1700 nm sensitivity
  • NIR-II contrast agent (e.g., SWNTs, rare-earth nanoparticles, or organic fluorophore)
  • Animal stabilization platform
  • Anesthesia system (isoflurane recommended)
  • Hair removal cream (for rodent imaging)
  • NIR excitation source (e.g., 808 nm laser with appropriate filters)
  • Data acquisition computer with imaging software

Procedure:

  • Contrast Agent Administration:

    • Prepare contrast agent according to manufacturer specifications.
    • Administer via tail vein injection (mouse) or other appropriate route.
    • For SWNT-based imaging, use doses of 1-10 nmol per animal in 100-200 μL saline [40].
    • Allow 5-60 minutes for circulation, depending on agent pharmacokinetics.

  • Animal Preparation:

    • Anesthetize animal using approved protocol (e.g., 2% isoflurane in oxygen).
    • Apply hair removal cream to imaging area and gently wipe clean after 1 minute.
    • Position animal on warming platform to maintain body temperature.
    • Secure in appropriate orientation for region of interest.

  • Imaging System Setup:

    • Cool InGaAs camera to operating temperature (-85°C for scientific grade).
    • Position excitation source at appropriate angle to minimize direct illumination of detector.
    • Set appropriate filters for excitation/emission wavelengths.
    • Configure acquisition software with desired parameters:
      • Exposure time: 50-500 ms (adjust based on signal intensity)
      • Frame rate: 1-10 fps for dynamic imaging
      • Binning: 2x2 for increased sensitivity if needed

  • Image Acquisition:

    • Acquire background image before contrast agent administration.
    • Capture time-series post-injection for dynamic processes.
    • For high-resolution static imaging, acquire multiple frames and average.
    • Maintain consistent illumination and camera settings throughout experiment.

  • Data Processing:

    • Subtract background fluorescence from all images.
    • Apply flat-field correction if illumination is non-uniform.
    • For quantitative analysis, generate standard curves using reference phantoms.
    • Calculate signal-to-background ratios in regions of interest.

Troubleshooting Notes:

  • Poor signal may require increased exposure time or contrast agent dose.
  • High background may indicate insufficient filter blocking of excitation light.
  • Motion artifacts may require deeper anesthesia or faster acquisition.

Protocol: AR Surgical Navigation System Validation

This protocol outlines the validation procedure for augmented reality surgical navigation systems integrated with NIR-II imaging data [41].

Materials Required:

  • AR display device (HoloLens or tablet with camera)
  • Registration markers or fiducials
  • Phantom model with known anatomy
  • Tracking system (optical or electromagnetic)
  • Segmentation software (e.g., U-Net based algorithm)
  • NIR-II imaging data set (preoperative)

Procedure:

  • System Calibration:

    • Calibrate AR display device according to manufacturer specifications.
    • Verify alignment of virtual and physical spaces using calibration patterns.
    • Test registration accuracy with known phantom models.

  • Data Integration:

    • Import preoperative NIR-II imaging data (DICOM format).
    • Apply segmentation algorithm to identify target structures.
    • Generate 3D model of surgical target, entry points, and critical structures.
    • Establish coordinate transformation between imaging data and physical space.

  • Registration Accuracy Assessment:

    • Position phantom model in surgical field.
    • Perform "virtual object automatic scanning" registration.
    • Measure discrepancy between virtual and physical landmarks.
    • Verify accuracy meets minimum requirement of 1 ± 0.1 mm [41].

  • Navigation Workflow Testing:

    • Practice surgical trajectory planning using AR guidance.
    • Verify dynamic auxiliary line for incision angle and depth.
    • Test system responsiveness to instrument movement.
    • Validate visualization of target-Entry point connection.

  • Clinical Validation:

    • Conduct pilot procedures in appropriate animal models.
    • Assess system performance by experienced surgeons.
    • Collect quantitative metrics: procedure time, accuracy, success rate.
    • Refine system based on clinical feedback.

G NIRLight NIR Excitation Light Source Tissue Biological Tissue with Deep Target NIRLight->Tissue Reduced scattering ContrastAgent NIR-II Contrast Agent Injection ContrastAgent->Tissue Emission NIR-II Emission Collection Tissue->Emission Deep penetration Detection InGaAs Camera Detection Emission->Detection High QE detection DataProcessing Image Processing & Analysis Detection->DataProcessing

Diagram 2: NIR-II Imaging Signal Path. This diagram illustrates the complete pathway from excitation to data processing, highlighting key advantages including reduced scattering and deep penetration.

Applications in Preclinical Research and Drug Development

The combination of advanced NIR-II imaging instrumentation and augmented reality navigation systems has enabled diverse applications across biomedical research and clinical translation. In drug development, NIR-II imaging provides unparalleled capability for monitoring drug distribution, pharmacokinetics, and treatment response in live animal models. The ability to track fluorescently-labeled therapeutic compounds in real-time through thick tissues offers significant advantages over traditional endpoint measurements [33].

In oncology research, NIR-II imaging enables detailed visualization of tumor vasculature, microenvironment, and heterogeneity. The high spatial and temporal resolution allows researchers to monitor dynamic processes such as drug uptake, tumor perfusion, and metastatic progression. When combined with multiplexed imaging approaches, researchers can simultaneously track multiple cell populations or molecular targets, providing comprehensive understanding of complex biological systems [1].

For neuroscience applications, NIR-II imaging facilitates non-invasive assessment of brain structure and function through the intact skull, overcoming the scattering that limits visible light imaging [39]. This capability is particularly valuable for longitudinal studies of disease progression, neurodevelopment, and therapeutic intervention in small animal models.

The integration of these imaging capabilities with augmented reality guidance systems creates powerful platforms for translational research. Surgeons can leverage deep-tissue information provided by NIR-II imaging to guide precise interventions, while researchers gain valuable insights into disease pathology and treatment mechanisms. This synergy between diagnostic imaging and therapeutic intervention represents a significant advancement in precision medicine.

Application Note: High-Resolution Vascular Imaging and Dynamics

Background and Principle

The second near-infrared window (NIR-II, 1000–1700 nm) fluorescence imaging leverages reduced photon scattering, minimal tissue autofluorescence, and lower light absorption in biological tissues compared to visible and NIR-I windows. This results in superior penetration depth (up to 20 mm) and micron-level spatial resolution, enabling unparalleled real-time visualization of deep-tissue vascular architectures and dynamic blood flow patterns [35] [32]. This application is foundational for studying cardiovascular diseases, tumor angiogenesis, and cerebrovascular dynamics.

Key Experimental Findings and Data

NIR-II imaging allows for precise quantification of vascular parameters. The following table summarizes key performance metrics for vascular imaging using different NIR-II probes:

Table 1: Performance of NIR-II Fluorophores in Vascular Imaging

Fluorophore Type Excitation/Emission (nm) Spatial Resolution Penetration Depth Key Vascular Application
SWCNTs (PEGylated) [35] N/A, Broad NIR-II emission ~100 µm Deep tissue imaging First-generation probe for vascular imaging
Agâ‚‚S Quantum Dots [35] Tunable 687-1294 nm High (micron-level) Several millimeters Non-specific tumor detection and vascular mapping
Biomimetic NIR-II FPs (HSA@CO-1080) [8] 1044/1079 nm High temporal-spatial resolution Enabled by 1064 nm excitation High-contrast lymphography and angiography
Organic Small Molecules (LS series) [43] Up to 1218 nm emission High Favorable for BBB penetration Visualization of brain vasculature and pathologies

Detailed Experimental Protocol: NIR-II Vascular Angiography

Objective: To perform real-time, high-resolution imaging of the vascular system in a live mouse model.

Materials:

  • Animal Model: Anesthetized mouse (e.g., C57BL/6).
  • Imaging System: NIR-II imaging setup (e.g., IR VIVO or LightIR system with an InGaAs camera) [26].
  • Fluorophore: 100 µL of IR-1048 dye or HSA@CO-1080 biomimetic fluorescent protein (1-2 nmol in PBS) [8] [26].
  • Anesthesia: Isoflurane vaporizer.
  • Software: Image acquisition and analysis software (e.g., MATLAB, ImageJ).

Procedure:

  • Animal Preparation: Anesthetize the mouse using 2-3% isoflurane and maintain anesthesia at 1-2% during imaging. Secure the animal in a supine position on a heated stage.
  • Tail Vein Cannulation: Gently warm the tail with a heat lamp to dilate the vein. Insert a 30-gauge insulin needle and secure it for intravenous (IV) injection.
  • System Setup: Configure the NIR-II imaging system with a 1064 nm laser for excitation (for HSA@CO-1080) or an 808 nm laser for other dyes. Set appropriate long-pass filters (e.g., LP1250 or LP1500) for emission collection [8].
  • Background Acquisition: Acquate a pre-injection image to serve as a background reference.
  • Fluorophore Administration: Inject the 100 µL bolus of the NIR-II probe via the tail vein catheter. Flush with saline.
  • Image Acquisition: Initiate continuous video-rate imaging immediately after injection. Capture data for 10-20 minutes to monitor the first pass and subsequent circulation of the probe.
  • Data Analysis:
    • Perfusion Analysis: Calculate time-to-peak and flow rate in regions of interest (ROIs) over major organs.
    • Vascular Morphometry: Use skeletonization algorithms to quantify vessel diameter, density, and branching points.

Troubleshooting Tips:

  • Ensure stable anesthesia to prevent motion artifacts.
  • Optimize laser power and exposure time to maximize signal-to-background ratio while avoiding detector saturation or tissue damage.
  • For deep-tissue imaging, the 1000-1350 nm range is optimal due to the trade-off between reduced scattering and low water absorption [32].

Application Note: Delineation of Tumor Margins and Microenvironment

Background and Principle

The chaotic tumor vasculature and dysregulated pathophysiology of the tumor microenvironment (TME) create opportunities for passive accumulation (Enhanced Permeability and Retention effect) and active targeting of NIR-II probes [32]. This allows for precise delineation of primary and metastatic tumors, visualization of the TME, and guidance for surgical resection with high signal-to-background ratios (SBR) [35] [26].

Key Experimental Findings and Data

NIR-II probes have been successfully applied across various cancer models. The table below compares different probes used in tumor imaging:

Table 2: NIR-II Probes in Tumor Imaging and Image-Guided Surgery

Probe Type / Strategy Target / Mechanism Key Finding Potential Clinical Utility
Au NCs-Pt Nanodrug [35] GSH Scavenging & Pt Delivery Visualized Pt transport in deep tumors via NIR-II; sensitized cells to chemo Theranostic platform for chemotherapy
RENPs@Lips [35] Passive Accumulation Double emission (1064/1345 nm); favorable biocompatibility & excretion Preclinical evaluation of physiological/pathological processes
Organic Small Molecules [32] EPR effect; some are targetable Fast clearance, superior biocompatibility; Milestone: ICG used in patient liver cancer surgery [32] Most promising for clinical translation in guided surgery
Clinical LightIR System [26] N/A (Imaging System) Resolved ~250 µm features; detected ICG to depths ≥4 mm under ambient light Back-table tumor margin assessment during surgery

Detailed Experimental Protocol: NIR-II Image-Guided Tumor Resection

Objective: To intraoperatively delineate primary tumor margins and identify microscopic metastatic deposits in a preclinical model.

Materials:

  • Animal Model: Mouse with a subcutaneously implanted tumor (e.g., 4T1 breast carcinoma).
  • Imaging System: Clinical-grade NIR-II system such as LightIR for open-field imaging or a preclinical IR VIVO system [26].
  • Fluorophore: 100 µL of a targeted organic small-molecule NIR-II probe (e.g., LS series, <500 Da) or a passively accumulating probe like Agâ‚‚S QDs via IV injection [32] [43].
  • Surgical Tools: Microsurgical instrument kit.

Procedure:

  • Probe Administration: 24 hours before surgery, inject the NIR-II probe intravenously to allow for sufficient accumulation and background clearance.
  • Pre-operative Imaging: Anesthetize the animal and perform a whole-body NIR-II scan to locate the primary tumor and potential metastases.
  • Surgical Exposure: Make a skin incision to expose the primary tumor under normal white light.
  • Intraoperative Imaging: Switch the LightIR system to NIR-II mode under ambient lighting. Identify the bright fluorescent signal defining the tumor boundary.
  • Resection Guidance: Perform the primary resection, leaving a small margin of suspected tissue.
  • Back-Table Margin Assessment: Excise the tumor and image the resected mass and the tumor bed on the LightIR system. The tumor bed should show minimal residual fluorescence, while the resected specimen should show a clear, continuous fluorescent rim.
  • Validation: Any fluorescent spots in the tumor bed indicate potential residual disease for further resection. Collect tissue samples for histopathological validation.

Troubleshooting Tips:

  • The optimal tumor-to-background ratio is often achieved 24-48 hours post-injection; a kinetic study is recommended for a new probe.
  • For the LightIR system, utilize its real-time background subtraction feature to enhance contrast in an open surgical field [26].

Application Note: Visualizing Immunological Interactions

Background and Principle

Understanding the dynamic interactions between immune cells and tumor cells within the TME is critical for developing immunotherapies. Intravital Microscopy (IVM) powered by NIR-II and Fluorescence Lifetime Imaging Microscopy (FLIM) enables real-time visualization of these interactions at single-cell resolution [44]. FLIM-FRET is particularly powerful for quantifying molecular interactions, such as those between immune checkpoint proteins (e.g., PD-1/PD-L1), which is a better predictor of immunotherapy response than simple protein expression levels [45].

Key Experimental Findings and Data

Advanced imaging techniques are revealing the dynamics of the immune response.

Table 3: Advanced Imaging Techniques for Immunological Studies

Technique Measured Parameter Key Application in Immunology Research Finding
FLIM-FRET [45] Fluorescence lifetime change due to energy transfer Quantifying PD-1/PD-L1 protein interaction Interaction level, not just expression, correlates with ICI efficacy [45]
Intravital Microscopy (IVM) [44] Real-time cell motility, migration, and interactions Visualizing T cell – tumor cell interactions in live animals Enables study of immune cell infiltration, killing, and evasion
FLIM Flow Cytometry [46] Fluorescence lifetime of single cells in flow High-throughput analysis of tumor cell heterogeneity Distinguished subpopulations in rat glioma; captured drug-induced nuclear changes

Detailed Experimental Protocol: FLIM-FRET for Immune Checkpoint Interaction

Objective: To quantify the interaction between PD-1 and PD-L1 in a live cell co-culture system using FLIM-FRET.

Materials:

  • Cells: Jurkat T-cells (or primary T-cells) expressing PD-1 tagged with a donor fluorophore (e.g., GFP).
  • Target Cells: Tumor cells expressing PD-L1 tagged with an acceptor fluorophore (e.g., RFP).
  • Imaging System: Multiphoton microscope equipped with FLIM capability.
  • Controls: Cells expressing donor-only and acceptor-only constructs.

Procedure:

  • Sample Preparation: Mix the donor-labeled and acceptor-labeled cells at an appropriate ratio on a glass-bottom dish. Allow cells to settle and interact.
  • FLIM Acquisition: Place the dish on the microscope stage. Focus on interacting cell pairs.
    • Use a two-photon laser (e.g., 920 nm) to excite the donor fluorophore (GFP).
    • Acquire fluorescence lifetime images of the donor channel.
  • Data Analysis:
    • Lifetime Calculation: Fit the fluorescence decay curve of the donor fluorophore in each pixel to obtain its lifetime (Ï„).
    • FRET Efficiency: Calculate the FRET efficiency (E) in regions of cell-cell contact using the formula: E = 1 - (Ï„DA / Ï„D), where Ï„DA is the donor lifetime in the presence of the acceptor, and Ï„D is the donor lifetime alone.
    • A significant decrease in Ï„DA compared to Ï„D indicates molecular interaction.
  • Inhibition Assay: Repeat the experiment after adding a therapeutic anti-PD-1 antibody (e.g., Pembrolizumab). The loss of FRET (lifetime returns to Ï„D) confirms the specific disruption of the PD-1/PD-L1 interaction [45].

Troubleshooting Tips:

  • Ensure optimal expression levels of donor and acceptor tags to avoid artifacts.
  • Carefully account for spectral bleed-through using control samples.
  • The interaction is transient; therefore, acquire images at a sufficient temporal resolution to capture binding events.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key Reagents and Materials for NIR-II and Intravital Imaging

Item Name Function / Description Example Use Case
NIR-II Organic Small Molecules [32] [43] Low molecular weight (<500 Da) fluorophores with tunable emission up to 1200+ nm; favorable pharmacokinetics. Vascular imaging, tumor targeting, and brain imaging due to potential BBB penetration.
Biomimetic NIR-II Fluorescent Proteins [8] Synthetic dyes (e.g., CO-1080) covalently bound to serum albumin (HSA), enhancing brightness and photostability. Creating long-circulating probes for high-contrast lymphography and angiography.
Rare-Earth-Doped Nanoparticles (RENPs) [35] Inorganic nanoparticles with large Stokes shifts, narrow emissions, and minimal photobleaching. Multiplexed imaging with distinct emission wavelengths (e.g., 1064 nm & 1345 nm).
Clinical LightIR Imaging System [26] Compact InGaAs camera-based system for real-time NIR-II imaging under ambient light. Intraoperative guidance and back-table assessment of tumor margins in a clinical setting.
FLIM Flow Cytometer [46] High-throughput system combining flow cytometry with FLIM, analyzing >10,000 cells/second. Rapid, label-free analysis of tumor cell heterogeneity and drug response based on metabolic states.
EpiaschantinColchicine Derivative 5-[(4r)-4-(3,4,5-Trimethoxyphenyl)tetrahydro-1h,3h-furo[3,4-c]furan-1-yl]-1,3-benzodioxoleHigh-purity 5-[(4r)-4-(3,4,5-trimethoxyphenyl)tetrahydro-1h,3h-furo[3,4-c]furan-1-yl]-1,3-benzodioxole for research. For Research Use Only. Not for human or veterinary use.
Rg3039Rg3039, CAS:1466525-84-7Chemical Reagent

Experimental Workflow and Signaling Pathways

NIR-II Fluorophore Design Strategy

The following diagram illustrates the strategic concepts behind designing advanced NIR-II fluorophores, moving beyond traditional methods.

G Start Goal: Redshift Emission for NIR-II Imaging Traditional Reduce HOMO-LUMO Band Gap Start->Traditional Conventional Coulomb Reduce Coulomb Attraction (Ec) Start->Coulomb Novel Strategy TradResult Increased Molecular Weight Potential Instability Traditional->TradResult Result CoulombResult Enhanced ICT Effect Longer Emission Wavelength Low Molecular Weight Coulomb->CoulombResult Result App2 Large, Complex Probes TradResult->App2 Limits App1 Small-Molecule Probes (e.g., LS Series Dyes) CoulombResult->App1 Enables

FLIM-FRET for Immune Checkpoint Analysis

This diagram outlines the workflow and principle of using FLIM-FRET to quantify immune checkpoint protein interactions.

G A 1. Label Proteins A1 PD-1 fused to Donor Fluorophore A->A1 A2 PD-L1 fused to Acceptor Fluorophore A->A2 B 2. Cells Interact A1->B A2->B C 3. Acquire FLIM Data B->C C1 Donor Lifetime (Ï„D) No Interaction C->C1 C2 Donor Lifetime (Ï„DA) Interaction (Ï„DA < Ï„D) C->C2 D 4. Calculate FRET Efficiency C1->D C2->D E High FRET Efficiency Indicates Strong Interaction D->E E = 1 - (Ï„DA / Ï„D) F 5. Therapeutic Intervention E->F F1 Add Anti-PD-1 Antibody F->F1 G FRET Signal Lost Lifetime Recovers to Ï„D F1->G Blocks Interaction

Fluorescence-guided surgery (FGS) and image-guided drug delivery (IGDD) represent transformative paradigms in precision medicine. The core of this advancement lies in the utilization of the second near-infrared window (NIR-II, 1000–1700 nm), which offers superior deep-tissue imaging capabilities compared to traditional visible light (400–700 nm) and the first near-infrared window (NIR-I, 700–900 nm) [4] [31]. NIR-II light experiences significantly reduced absorption, scattering, and autofluorescence from biological tissues, enabling deeper tissue penetration (5–20 mm) and higher signal-to-background ratios (SBR) for high-contrast imaging [18] [1]. This review details specific application notes and experimental protocols for leveraging NIR-II fluorescence imaging in surgical guidance and targeted drug delivery, providing a practical toolkit for researchers and drug development professionals.

Application Note 1: NIR-II Fluorescence-Guided Oncologic Surgery

Rationale and Principle

The principal challenge in curative cancer surgery is the complete resection of malignant tissue while preserving healthy structures. NIR-II FGS addresses this by providing real-time, high-resolution visualization of tumors, vasculature, and critical anatomy. The enhanced penetration and reduced scattering of NIR-II light allow surgeons to distinguish sub-millimeter tumor margins and micrometastases that are otherwise invisible to the naked eye or under NIR-I imaging [4] [47]. This is particularly valuable for tumors located in highly vascularized or anatomically complex regions.

Quantitative Comparison of NIR Windows

The table below summarizes the key advantages of NIR-II imaging over other optical windows.

Table 1: Quantitative Comparison of Optical Imaging Windows for Biomedical Applications

Imaging Parameter Visible (400-700 nm) NIR-I (700-900 nm) NIR-II (1000-1700 nm)
Tissue Penetration Depth Shallow (1-2 mm) Moderate (1-5 mm) Deep (5-20 mm) [1]
Tissue Autofluorescence Very High High Negligible/Low [31]
Photon Scattering Very High High Reduced [18]
Spatial Resolution Low Moderate High (up to ~25 μm) [4]
Signal-to-Background Ratio (SBR) Low Moderate High [31]

Experimental Protocol: NIR-II Guided Tumor Resection

Objective: To precisely resect a solid tumor in a murine model using a NIR-II fluorescent probe for intraoperative guidance.

Materials:

  • NIR-II Fluorescent Probe: e.g., Ag2S Quantum Dots (QDs) [31] or a targeted organic dye (e.g., IRDye800CW-conjugated to Cetuximab) [48].
  • Animal Model: Immunocompromised mouse (e.g., BALB/C nude) with a subcutaneously or orthotopically implanted tumor.
  • NIR-II Imaging System: Camera with an InGaAs detector, NIR-II compatible lenses, and appropriate laser excitation sources (e.g., 808 nm or 980 nm) [31] [1].
  • Software: For image acquisition and analysis (e.g., ImageJ with custom macros).

Procedure:

  • Probe Administration: Intravenously inject the NIR-II probe (e.g., 100-200 µL of 100 µM solution) via the tail vein. For targeted probes, allow 24-48 hours for systemic clearance and specific accumulation at the tumor site [48].
  • Pre-operative Imaging: Anesthetize the mouse and place it in the NIR-II imaging system. Acquire a baseline NIR-II fluorescence image to confirm tumor localization and define the initial tumor margin.
  • Surgical Resection:
    • Perform the surgery under sterile conditions.
    • Use the NIR-II imaging system in real-time to guide the resection. The tumor will be highlighted with a strong fluorescent signal.
    • Periodically image the surgical field to check for residual fluorescent tissue.
  • Assessment of Resection Completeness:
    • After putative complete resection, image the tumor bed to check for any residual fluorescent signal.
    • Resect any remaining fluorescent spots.
  • Ex Vivo Analysis:
    • Image the resected tumor mass to confirm fluorescence.
    • Collect and image major organs (liver, spleen, kidneys) to study the biodistribution and clearance of the probe.
  • Data Analysis:
    • Calculate the Signal-to-Background Ratio (SBR) as: SBR = (Mean Intensity of Tumor) / (Mean Intensity of Adjacent Normal Tissue).
    • Use software to quantify the fluorescence intensity in the tumor bed pre- and post-resection to objectively assess the completeness of surgery.

Application Note 2: NIR-II Image-Guided Drug Delivery for Cancer Theranostics

Rationale and Principle

Image-guided drug delivery (IGDD) systems merge diagnostic capability with therapeutic intervention, forming a "theranostic" platform. NIR-II fluorescence is ideally suited for monitoring the pharmacokinetics and pharmacodynamics of drug carriers in real-time [18]. This allows researchers to non-invasively track the accumulation of a drug-loaded nanocarrier in a tumor, monitor its distribution within the tumor microenvironment, and even trigger drug release at the desired site, thereby optimizing the therapeutic index and minimizing off-target effects [18] [49].

The Scientist's Toolkit: Key Reagents for NIR-II IGDD

The table below lists essential materials for constructing a NIR-II IGDD system.

Table 2: Research Reagent Solutions for NIR-II Image-Guided Drug Delivery

Item Function/Description Examples
NIR-II Fluorophore Serves as the imaging agent; can be conjugated to or encapsulated within a drug carrier. Organic dyes (e.g., CH-4T [4]), Ag2S Quantum Dots [31], Lanthanide-doped Nanoparticles (DCNPs) [1].
Drug Nanocarrier Carries the therapeutic payload; can be engineered for targeted delivery. Liposomes, Polymeric Nanoparticles (e.g., PLGA), Dendrimers, Extracellular Vesicles [18].
Targeting Moiety Directs the nanocarrier to specific biomarkers on cancer cells. Antibodies (e.g., anti-EGFR), Peptides (e.g., cRGD), Folic Acid [18] [48].
Stimuli-Responsive Material Enables controlled drug release in response to specific triggers. pH-sensitive polymers, ROS-cleavable linkers, light-activatable coatings [4].
NIR-II Imaging System For in vivo, real-time tracking of the theranostic agent. InGaAs camera, NIR-II laser excitations (808, 980 nm), spectral filters [31] [1].
DBCO-PEG8-acidDBCO-PEG8-acid|Bifunctional PEG Linker for Click Chemistry
MIDD0301MIDD0301

Experimental Protocol: Tracking Drug Carrier Delivery and Release

Objective: To visualize the accumulation of a NIR-II-labeled drug carrier in a tumor and assess its drug release profile.

Materials:

  • Theranostic Nanoparticle: e.g., a biodegradable polymer nanoparticle (like PLGA) loaded with a chemotherapeutic drug (e.g., Doxorubicin) and co-encapsulated with Ag2S QDs or an organic NIR-II dye.
  • Animal Model: Mouse model with a window chamber tumor or a subcutaneous tumor.
  • NIR-II Imaging System: As in Protocol 2.3.

Procedure:

  • Systemic Administration: Inject the theranostic nanoparticle intravenously into the tumor-bearing mouse.
  • Real-Time Pharmacokinetic Imaging:
    • Acquire sequential NIR-II images at set time points (e.g., 5 min, 30 min, 1 h, 4 h, 24 h) post-injection.
    • Monitor the circulation of the nanoparticle and its gradual accumulation in the tumor via the Enhanced Permeability and Retention (EPR) effect or active targeting.
  • Triggered Drug Release (Optional): If the nanoparticle is designed for triggered release (e.g., via photothermal effect), apply the external stimulus (e.g., NIR laser) at the time of peak tumor accumulation.
  • Image Analysis:
    • Quantify Tumor Accumulation: Draw a region of interest (ROI) around the tumor and a background ROI on normal muscle tissue. Plot a time-activity curve of the fluorescence intensity in the tumor to determine the time to peak accumulation [50].
    • Assess Penetration: For high-resolution systems, analyze the distribution of the fluorescence signal within the tumor core versus the periphery.

Visualization of NIR-II Phototheranostic Workflows

The following diagrams, generated using DOT language, illustrate the core concepts and experimental workflows described in this application note.

NIR-II Phototheranostic Cycle

This diagram illustrates the integrated "diagnose-treat-monitor" cycle enabled by NIR-II theranostic probes.

G Start 1. Systemic Injection of NIR-II Theranostic Probe A 2. Probe Accumulates in Tumor Tissue Start->A B 3. NIR-II Fluorescence Imaging Precise Tumor Delineation A->B C 4a. Guided Surgical Resection B->C D 4b. Triggered Therapy (PTT/PDT/Drug Release) B->D E 5. Real-time Monitoring of Treatment Efficacy C->E D->E E->B Feedback Loop

NIR-II IGDD Experimental Workflow

This flowchart outlines the key steps for a typical NIR-II image-guided drug delivery experiment, from formulation to analysis.

G A Formulate Theranostic Agent (Drug + NIR-II Fluorophore + Carrier) B Administer Intravenously in Tumor Model A->B C Real-time NIR-II Imaging to Track Biodistribution B->C D Quantify Tumor Accumulation and Kinetics C->D E Apply Release Stimulus (e.g., NIR Laser) D->E F Monitor Drug Release & Therapeutic Response E->F

The integration of NIR-II fluorescence imaging with surgical guidance and targeted drug delivery systems marks a significant leap forward in theranostics. The protocols and application notes provided here offer a foundational framework for researchers to implement these cutting-edge technologies. As the field progresses, overcoming challenges in probe biocompatibility, complex synthesis, and clinical translation through innovative molecular design and interdisciplinary collaboration will be crucial [4]. The future of precision medicine lies in these multi-functional platforms that allow for simultaneous diagnosis, treatment, and monitoring, ultimately leading to improved patient outcomes.

Overcoming Technical Hurdles: Strategies for Enhancing Fluorophore Performance and Imaging Fidelity

Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) has emerged as a transformative biomedical imaging modality, offering superior spatial resolution and tissue penetration compared to traditional NIR-I imaging [51] [52]. This enhanced performance stems from significantly reduced photon scattering, lower tissue autofluorescence, and diminished light absorption in biological tissues within this spectral region [52] [53]. However, the development of high-performance NIR-II fluorophores has been persistently hampered by the quantum yield bottleneck – the inherent tendency of fluorophores with extended π-conjugation systems to exhibit low fluorescence efficiency due to increased non-radiative decay pathways [37] [54].

The quantum yield (QY) of a fluorophore, defined as the ratio of photons emitted to photons absorbed, directly determines imaging brightness and consequently impacts detection sensitivity, signal-to-noise ratio, and ultimately, diagnostic accuracy [54]. For NIR-II organic fluorophores, quantum yields typically range from 0.01% to 3.89%, significantly lower than their visible-light counterparts [54]. This limitation has catalyzed intensive research into molecular engineering strategies designed to enhance the brightness of NIR-II fluorophores while maintaining their deep-tissue penetration capabilities, forming a critical frontier in biomedical optics research [55] [37].

Molecular Engineering Strategies for Brightness Enhancement

Rigidification of Molecular Structure

A primary approach to enhancing quantum yield involves reducing non-radiative decay by imposing structural rigidity on fluorophore systems. The introduction of rigid cyclohexanol structures at the center of cyanine/polymethine dyes restricts molecular vibration and rotation, key contributors to non-radiative energy loss [37]. Similarly, engineering increased dihedral angles in donor-acceptor-donor (D-A-D) architectures through the incorporation of rigid alkyl thiophene moieties has demonstrated significant quantum yield improvements by minimizing collisional quenching [37].

Table 1: Structural Rigidification Strategies and Their Impact on Quantum Yield

Engineering Strategy Molecular Target Mechanism of Action Reported QY Improvement
Cyclohexanol Incorporation Cyanine/Polymerhine Dyes Restricts molecular vibration & rotation ~2-3 fold increase [37]
Dihedral Angle Control D-A-D Structured Dyes Minimizes collisional quenching Significant enhancement [37]
Aggregation-Induced Emission (AIE) AIE Luminogens Restricts intramolecular rotation Dramatic fluorescence enhancement [56]

Shielding Group Incorporation

Molecular shielding represents another powerful strategy for quantum yield enhancement. The incorporation of shielding units (S) containing long polyethylene glycol (PEG) groups or dialkoxy-substituted benzene/fluorene-based moieties in S-D-A-D-S architectures creates a protective microenvironment around the fluorophore core [37]. These shielding groups function through multiple mechanisms: they improve molecular dispersion in aqueous solutions, reduce π-π stacking-induced quenching, and create steric hindrance that minimizes collisions with quenching species such as water molecules and dissolved oxygen [37] [54]. The strategic placement of these shielding units is critical to their efficacy, as they must provide protection without disrupting the electronic transitions responsible for fluorescence.

Protein-Based Fluorophore Chaperoning

An innovative bioengineering approach involves using recombinant proteins as molecular chaperones to enhance fluorophore brightness. Research has identified that Domain III (DIII) of human serum albumin serves as a high-affinity binding pocket for cyanine dyes [37]. When fluorophores are encapsulated within this hydrophobic domain, they experience multiple benefits: stabilization of the excited state, reduced non-radiative decay through restricted molecular motion, and protection from collisional quenching [37]. This protein-dye complex formation can enhance fluorescence brightness by approximately 1.6-fold compared to free dye [37]. Furthermore, this strategy allows genetic editing to tune complex size and pharmacokinetics while providing a biocompatible coating that improves biosafety and clinical translation potential [37].

G Start Quantum Yield Limitation in NIR-II Fluorophores Strat1 Molecular Rigidification Start->Strat1 Strat2 Shielding Group Incorporation Start->Strat2 Strat3 Protein-Based Chaperoning Start->Strat3 Mech1 Reduced vibrational/ rotational freedom Strat1->Mech1 Mech2 Steric protection from quenchers Strat2->Mech2 Mech3 Hydrophobic pocket stabilization Strat3->Mech3 Outcome Enhanced Quantum Yield & Brightness Mech1->Outcome Mech2->Outcome Mech3->Outcome

Extended π-Conjugation with Heteroatom Substitution

Systematic extension of π-conjugation systems combined with strategic heteroatom substitution has proven effective in bathochromically shifting absorption and emission while maintaining reasonable quantum yields. The incorporation of dicyanomethylene-4H-benzopyran (DCMO) and its heteroatom variants (DCMS with sulfur, DCMSe with selenium) as electron-withdrawing acceptors in conjunction with extended polymethine bridges enables emission tuning up to 1060 nm while preserving fluorescence efficiency [57]. Heavier chalcogen atoms (S, Se) promote greater electronic delocalization and narrower HOMO-LUMO gaps, enabling longer wavelength emissions. However, this approach must be carefully balanced against the "cyanine limit" – the point at which further conjugation leads to symmetry breaking and fluorescence quenching [54].

Table 2: Photophysical Properties of Representative Engineered NIR-II Fluorophores

Fluorophore λabs/λem (nm) Quantum Yield (%) Brightness (M⁻¹ cm⁻¹) Key Structural Features
Flav7 1027/1053 0.610 1470.1 Rigidified heterocyclic terminals [54]
LZ-1105 1058/1100 3.890 5757.2 Optimized shielding groups [54]
HC-1222 1180/1222 0.016 18.8 Extended conjugation [54]
IR-783@DIII 783/~1000 Enhanced 1.6x Significantly increased Protein chaperoning [37]
CL-S3 ~1089/1060 N/A High SBR (43.5) Extended π-conjugation [57]

Experimental Protocols for Quantum Yield Evaluation

Relative Quantum Yield Measurement Protocol

Principle: This method determines fluorophore quantum yield relative to a reference standard with known quantum yield in the same solvent system [54].

Materials:

  • Spectrophotometer (UV-Vis-NIR)
  • NIR-II fluorescence spectrometer with InGaAs detector
  • Cuvettes suitable for NIR measurements
  • Reference standard (IR-26 in 1,2-dichloroethane, QY = 0.05%)
  • Solvents (high purity, spectroscopic grade)
  • Nitrogen gas for degassing

Procedure:

  • Prepare matched absorbance solutions (<0.1) of sample and reference in the same solvent to minimize inner filter effects.
  • Record absorption spectra of both solutions using spectrophotometer.
  • Record emission spectra using identical instrumental parameters (slit widths, detector gain).
  • Integrate the area under the fluorescence emission curves for both sample and reference.
  • Calculate quantum yield using the following equation: ΦX = ΦST × (AX/AST) × (ηX2/ηST2) Where Φ is quantum yield, A is integrated emission area, η is refractive index of solvent, and subscripts X and ST denote sample and standard, respectively.
  • Repeat measurements in triplicate and average results.

Technical Notes: Ensure complete degassing of solutions to eliminate oxygen quenching. Maintain low analyte concentrations to avoid aggregation effects. Account for solvent-dependent refractive index variations [54].

Protein-Fluorophore Complex Formation Protocol

Principle: This protocol describes the formation and characterization of protein-fluorophore complexes for brightness enhancement based on the albumin chaperoning strategy [37].

Materials:

  • Recombinant human serum albumin domains (DI, DII, DIII)
  • Cyanine dyes (IR-783, ICG, IR-1061, etc.)
  • Phosphate buffered saline (PBS), pH 7.4
  • Size exclusion chromatography columns
  • SDS-PAGE equipment
  • NIR-II imaging system

Procedure:

  • Prepare stock solutions of albumin domains (100 μM) in PBS.
  • Prepare cyanine dye stock solution (1 mM) in DMSO.
  • Mix protein and dye at molar ratios ranging from 1:1 to 1:3 in PBS.
  • Incubate mixtures at 37°C for 2 hours or 60°C for 10 minutes for complex formation.
  • Purify complexes using size exclusion chromatography to remove unbound dye.
  • Verify complex formation using SDS-PAGE with NIR-II fluorescence detection.
  • Characterize optical properties using absorption and emission spectroscopy.
  • Determine binding affinity using bio-layer interferometry (BLI) for Kd measurement.

Technical Notes: Optimal complex formation may require temperature optimization. Free dye must be completely removed for accurate quantum yield determination. Binding affinity varies significantly among dye structures – DiR shows notably weak binding (Kd = 144 μM) compared to IR-783 (Kd = 0.57 nM with DIII) [37].

G Start Protein-Fluorophore Complex Preparation Step1 Prepare albumin domain solutions (100 μM in PBS) Start->Step1 Step2 Prepare cyanine dye stock (1 mM in DMSO) Step1->Step2 Step3 Mix protein & dye (1:1 to 1:3 molar ratio) Step2->Step3 Step4 Incubate: 37°C/2h or 60°C/10min Step3->Step4 Step5 Purify via size exclusion chromatography Step4->Step5 Step6 Verify complex formation (SDS-PAGE + NIR detection) Step5->Step6 Step7 Characterize optical properties Step6->Step7 Output Characterized Protein- Fluorophore Complex Step7->Output

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents for NIR-II Fluorophore Development

Reagent/Material Function/Application Specific Examples Key Characteristics
Cyanine Dyes NIR-II fluorophore core structures IR-783, ICG, IR-1061, IR-1048, Cy7, Cy7.5 [37] [54] Tunable emission, high extinction coefficients
Recombinant Albumin Domains Fluorophore chaperoning for brightness enhancement Domain III (DIII) of HSA [37] High-affinity dye binding (Kd = 0.57 nM for IR-783)
Shielding Groups Reduce collisional quenching PEG groups, dialkoxy-substituted benzene/fluorene [37] Steric hindrance, improved solubility
Heterocyclic Terminal Moieties Bathochromic shift engineering DCMO, DCMS, DCMSe [57] Electron-withdrawing capability, heavy atom effect
Reference Standards Quantum yield determination IR-26 in DCE (QY = 0.05%) [54] Established reference for NIR-II QY measurements
Solvent Systems Photophysical characterization DCM, DCE, MeOH, DMSO, PBS [54] Varying polarity for solvatochromism studies

The molecular engineering strategies outlined in this application note provide a robust toolkit for addressing the persistent quantum yield bottleneck in NIR-II fluorescence imaging. Through integrated approaches combining structural rigidification, steric shielding, protein chaperoning, and rational π-conjugation design, researchers can systematically enhance fluorophore brightness while maintaining the deep-tissue penetration advantages of the NIR-II window.

The ongoing development of these engineering strategies continues to push the boundaries of in vivo imaging capabilities. Recent innovations in direct NIR-II chemiluminescence platforms [57] and genetically engineered protein-fluorophore complexes [37] represent promising avenues that may circumvent traditional quantum yield limitations entirely. As these molecular engineering approaches mature, they will undoubtedly accelerate the clinical translation of NIR-II fluorescence imaging, ultimately enhancing diagnostic precision and therapeutic monitoring capabilities across a spectrum of disease states.

The experimental protocols and materials detailed herein provide a foundation for researchers to implement these brightness enhancement strategies in their own NIR-II fluorophore development workflows, contributing to the collective effort to overcome the quantum yield bottleneck in deep-tissue bioimaging.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) has emerged as a transformative modality for deep-tissue bioimaging, offering significant advantages over traditional visible (400–700 nm) and NIR-I (700–900 nm) imaging. The longer wavelengths in the NIR-II window experience reduced photon scattering, lower tissue autofluorescence, and decreased light absorption by chromogenic biomolecules, resulting in millimeter- to centimeter-level penetration depths with micrometer spatial resolution [58] [14] [51]. This exceptional performance has positioned NIR-II fluorescence imaging as an indispensable tool for preclinical research and clinical applications, particularly in cancer diagnosis, image-guided surgery, and therapeutic monitoring.

However, the benefits across the broad NIR-II spectrum are not uniform. The NIR-IIa (1000–1300 nm) and NIR-IIb (1500–1700 nm) sub-regions are particularly valuable for deep-tissue imaging due to an optimal trade-off between reduced scattering and water absorption [14] [32]. As water absorption peaks sharply around 1450 nm, imaging in the NIR-IIb window can leverage rising light absorption to attenuate scattering photons, thereby further suppressing background interference [36]. Consequently, developing bright fluorophores with emissions red-shifted beyond 1000 nm, particularly into the NIR-IIa and NIR-IIb regions, represents a critical frontier in advancing deep-tissue imaging capabilities. This application note details molecular design strategies, experimental protocols, and reagent solutions to achieve these red-shifted emissions for enhanced biomedical imaging.

Molecular Design Strategies for Red-Shifting Emission

Donor-Acceptor-Donor (D-A-D) Architectures and Donor Engineering

The D-A-D molecular architecture, featuring an electron-accepting (A) core symmetrically flanked by electron-donating (D) units, provides a highly tunable platform for achieving NIR-II emission.

  • Core Design Principle: The electron-withdrawing strength of the acceptor core directly influences the intramolecular charge transfer (ICT) character and thereby the emission wavelength. Strong acceptors such as benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole (BBTD) are widely used to achieve emissions beyond 1100 nm [36] [32].
  • Strategy of Reduced D-A Distance: A powerful strategy for red-shifting emission involves enhancing the donor strength while simultaneously reducing the physical distance between the donor and acceptor. This intensifies their electronic interaction, narrowing the energy gap between the highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO). As demonstrated with the 2FT-(o)CB molecule, this approach can achieve absorption peaks beyond 800 nm and emission maxima extending to 1215 nm [36].
  • Twisted Molecular Structures: Incorporating steric hindrance, such as ortho-positioned alkyl chains on Ï€-bridges, induces a twisted molecular conformation. This twisting inhibits detrimental Ï€-Ï€ stacking in the aggregated state, a common culprit for fluorescence quenching. Single-crystal evidence for dye 2FT-(o)CB confirms an ultralong molecular packing distance exceeding 8.5 Ã…, which effectively suppresses concentration quenching and maintains high brightness [36].

Cyanine Dye Modification

Cyanine dyes, characterized by a polymethine chain connecting two heterocyclic units, are another major class of NIR-II fluorophores.

  • Traditional Limitation: Simply extending the polymethine chain to red-shift emission often compromises photostability and fluorescence quantum yield (QY) [58].
  • Alternative Strategy: Modifying the terminal heterocyclic groups provides a viable path. For instance, replacing a sulfur atom with an oxygen atom and incorporating strong electron-donating dimethylamino groups has led to the development of flavylium-based fluorophores (e.g., Flav7) with high QYs [58].
  • Protein-Seeking Dyes: A novel approach involves designing cyanine-based "protein-seeking" dyes that covalently bind to endogenous proteins like human serum albumin (HSA). This binding event can restrict molecular rotation and reduce non-radiative decay, resulting in a significant fluorescence enhancement (e.g., 22-fold for HSA@CO-1080) and a red-shift in emission [8].

Aggregation-Induced Emission (AIE)

Engineering fluorophores with aggregation-induced emission (AIE) properties is particularly effective for designing bright nanofluorophores. AIE luminogens (AIEgens) are non-emissive in solution but become highly fluorescent in their aggregated state, which is ideal for nanoparticle formulation. Molecular design for NIR-II AIEgens focuses on creating twisted D-A-D structures full of molecular rotors. The restriction of intramolecular motion in the aggregate state blocks non-radiative pathways, forcing the energy to be released radiatively, thereby enhancing fluorescence in the NIR-II window [36].

Quantitative Data on Advanced NIR-II Fluorophores

The following tables summarize the photophysical properties of state-of-the-art NIR-II fluorophores developed using the strategies above, providing a comparative overview of their performance.

Table 1: Photophysical Properties of Representative D-A-D NIR-II Fluorophores

Molecule Name Acceptor Core Donor Unit Absorption Peak (nm) Emission Peak (nm) Molar Extinction Coefficient (M⁻¹cm⁻¹) Reference
2TT-(o)CB BBTD Triphenylamine-based alkylthiophene 695 ~1000 1.1 × 10⁴ [36]
2MTT-(o)CB BBTD Methoxy-triphenylamine 736 ~1050 1.3 × 10⁴ [36]
2MPT-(o)CB BBTD Methoxy-diphenylamine 860 ~1150 1.8 × 10⁴ [36]
2FT-(o)CB BBTD Fluorene-based diamine 829 1215 2.3 × 10⁴ [36]

Table 2: Performance of Other NIR-II Fluorophore Classes

Fluorophore Class/Name Type Absorption Peak (nm) Emission Peak (nm) Key Feature Reference
Flav7 Cyanine N/A >1000 QY of 0.53 [58]
HSA@CO-1080 Protein-seeking cyanine 1044 1079 22-fold fluorescence enhancement [8]
NDP (Activated) Naphthalene diimide 1066 1138, 1316 "Off-on-off" probe [59]

Experimental Protocols for Validation

Protocol: Evaluating Photophysical Properties of NIR-II Dyes

This protocol is essential for characterizing newly synthesized NIR-II fluorophores in solution [36] [59].

  • Sample Preparation: Prepare a stock solution of the purified dye in a high-purity organic solvent such as tetrahydrofuran (THF) or dimethyl sulfoxide (DMSO). Perform serial dilutions to achieve a final concentration suitable for spectroscopy (typically 1-10 µM).
  • UV-vis-NIR Absorption Spectroscopy:
    • Use a UV-vis-NIR spectrophotometer equipped with a PMT or InGaAs detector.
    • Blank the instrument with the pure solvent.
    • Place the diluted dye solution in a quartz cuvette (path length: 1 cm) and record the absorption spectrum from 500 nm to 1300 nm.
    • Calculate the molar extinction coefficient (ε) using the Beer-Lambert law (A = ε × c × l) from a dilution series of known concentrations.
  • Photoluminescence (PL) Spectroscopy:
    • Use a fluorescence spectrometer with a NIR-sensitive detector (e.g., liquid nitrogen-cooled InGaAs array).
    • Excite the sample at its maximum absorption wavelength. Common laser excitations include 808 nm, 980 nm, and 1064 nm.
    • Record the emission spectrum across the NIR-I and NIR-II windows (e.g., 900–1700 nm).
    • For quantum yield (QY) measurement, use a known NIR-II dye (e.g., IR-26 in 1,2-dichloroethane with a QY of 0.05%) as a reference, ensuring matched optical densities at the excitation wavelength.

Protocol: Nanoprecipitation for Formulating NIR-II Nanofluorophores

Most organic NIR-II dyes are hydrophobic and require nanoparticle formulation for biological applications [36] [59].

  • Organic Phase Preparation: Dissolve the NIR-II dye and an amphiphilic polymer (e.g., polystyrene-co-maleic anhydride, PSMA; or F-127 copolymer) in THF or DMSO at a defined mass ratio (e.g., 1:5 dye-to-polymer).
  • Aqueous Phase Preparation: Filter phosphate-buffered saline (PBS, 1X, pH 7.4) or pure water through a 0.22 µm filter.
  • Nanoprecipitation:
    • Rapidly inject the organic solution (e.g., 1 mL) into vigorously stirred aqueous solution (e.g., 10 mL) using a syringe pump or coaxial microfluidic mixer.
    • Continue stirring for 2-4 hours to allow for complete solvent evaporation.
  • Purification and Characterization:
    • Purify the resulting nanofluorophore suspension by centrifugal filtration to remove organic solvent and unencapsulated dye.
    • Wash several times with PBS.
    • Characterize the hydrodynamic diameter and zeta potential using dynamic light scattering (DLS).
    • Determine the concentration via fluorescence correlation or a BCA assay for the polymer component.

Protocol: Assessing In Vivo Imaging Performance

This protocol validates the deep-tissue imaging performance of the formulated NIR-II probes [36].

  • Animal Model: Use immunodeficient mice (e.g., BALB/c nude mice) bearing subcutaneous or orthotopic tumors.
  • Probe Administration: Inject the NIR-II nanofluorophores intravenously via the tail vein at an appropriate dose (e.g., 100-200 µL of ~100 µM dye concentration).
  • NIR-II Imaging Setup:
    • Use a NIR-II imaging system equipped with a 1064 nm laser for excitation and an InGaAs camera for detection.
    • Anesthetize the mouse and place it in the imaging chamber.
    • Set the laser power and camera integration time to sub-saturation levels. Use a series of long-pass filters (e.g., 1000 LP, 1100 LP, 1250 LP, 1400 LP, 1500 LP) to collect signals in different spectral windows.
  • Data Acquisition and Analysis:
    • Acquire time-lapse images post-injection to monitor probe distribution and clearance.
    • For deep-tissue contrast evaluation, image the mouse abdomen to visualize spine or deep tumors through tissue.
    • Quantify the signal-to-background ratio (SBR) by measuring the mean intensity in the target region (e.g., tumor) and a adjacent background tissue region.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for Developing and Testing Red-Shifted NIR-II Probes

Reagent / Material Function / Application Examples / Notes
BBTD Acceptor Core Strong electron-acceptor for D-A-D dyes; enables emission >1200 nm. Central unit in 2FT-(o)CB and related molecules [36].
Triphenylamine Donors Electron-donating units with tunable strength via substituents. Used in 2TT-(o)CB; methoxy groups enhance donating power [36].
PSMA Polymer Amphiphilic polymer for nanoprecipitation; provides colloidal stability. Polystyrene-co-maleic anhydride; forms small, stable nanoparticles [36].
F-127-D-Gal Polymer Functionalized copolymer for active targeting and biocompatibility. Pluronic F-127 conjugated with D-Galactose for liver-targeting [59].
Human Serum Albumin (HSA) Endogenous protein for creating biomimetic NIR-II fluorescent proteins. Covalently binds protein-seeking dyes, enhancing brightness & biocompatibility [8].
1064 nm Laser Optimal excitation source for long-wavelength NIR-II dyes. Deeper penetration than 808 nm; reduces tissue autofluorescence [8] [59].
InGaAs Camera Essential detector for NIR-II light. Requires cooling (e.g., liquid Nâ‚‚) for low-noise imaging in 1000-1700 nm range.

Strategic Workflow and Visualization

The following diagram illustrates the integrated workflow for developing and applying red-shifted NIR-II fluorophores, from molecular design to biological validation.

workflow Start Molecular Design Strategy A D-A-D Architecture (Strong Acceptor, Powerful Donor) Start->A B Cyanine Modification (Heterocycle Engineering) Start->B C AIEgen Design (Twisted Structures) Start->C D Chemical Synthesis & Purification A->D B->D C->D E In Vitro Characterization (Absorption/Emission, QY) D->E F Nanoparticle Formulation (Nanoprecipitation) E->F G In Vitro Validation (Cell uptake, Cytotoxicity) F->G H In Vivo NIR-II Imaging (Deep-tissue penetration, SBR) G->H End Application: Bioimaging & Image-Guided Surgery H->End

Diagram 1: Integrated R&D Workflow for Red-Shifted NIR-II Fluorophores

The strategic red-shifting of emission wavelengths into the NIR-IIa and NIR-IIb windows is paramount for unlocking the full potential of deep-tissue fluorescence imaging. The molecular design strategies outlined—primarily focused on intensifying donor-acceptor interactions in D-A-D architectures, engineering cyanine dyes, and leveraging AIE effects—provide a robust roadmap for developing brighter, longer-wavelength fluorophores. The accompanying experimental protocols and essential reagent toolkit offer a practical foundation for researchers to synthesize, characterize, and validate new NIR-II probes. As these strategies continue to evolve, they will accelerate the translation of NIR-II fluorescence imaging from a powerful research tool into a clinical reality for sensitive disease diagnosis and precise image-guided interventions.

Application Notes

The efficacy of fluorescence imaging agents, particularly those operating in the second near-infrared window (NIR-II, 900-1700 nm), is fundamentally governed by their pharmacokinetic (PK) profile. Optimizing this profile—specifically the balance between circulation time, target specificity, and renal clearance—is paramount for achieving high signal-to-background ratios (SBR) in deep-tissue imaging. The NIR-II window offers significant advantages over traditional NIR-I imaging, including reduced photon scattering, lower tissue autofluorescence, and deeper tissue penetration, but these benefits can only be fully leveraged with contrast agents possessing tailored pharmacokinetic properties [31] [36]. Effective PK optimization enables precise delineation of biological structures and disease targets, such as tumors, with reported SBR values exceeding 100 at tissue depths of 4-6 mm [36].

The Critical Interplay of PK Parameters

The core challenge in molecular probe design lies in managing the interplay of three key parameters:

  • Circulation Time: A sufficiently long circulation time is necessary for a contrast agent to extravasate, diffuse into the target tissue, and bind its molecular target. This is often a prerequisite for high target specificity.
  • Target Specificity: This refers to the agent's ability to accumulate at the site of interest (e.g., a tumor) while clearing from non-target tissues. Specificity is achieved through active targeting (e.g., antibody- or affibody-conjugation) or passive targeting (e.g., the Enhanced Permeability and Retention effect in tumors) [60] [61].
  • Renal Clearance: Rapid clearance of unbound agent from the systemic circulation is essential for reducing background signal and improving image contrast. Renal clearance is the process by which the kidneys remove a substance from the plasma, defined as the volume of plasma completely cleared of the substance per unit time [62].

Achieving an optimal balance requires a deep understanding of how a molecule's physicochemical properties—such as size, hydrophilicity, and molecular geometry—influence its pharmacokinetic pathway. For instance, a highly hydrophilic agent may exhibit rapid renal clearance, reducing background signal but potentially limiting its target accumulation time. Conversely, a more lipophilic agent may have a longer circulation time but suffer from high non-specific uptake in off-target tissues, leading to increased background noise [63].

Experimental Protocols

Protocol 1: Determining Renal Clearance of NIR-II Contrast Agents

Objective: To quantitatively determine the renal clearance of a NIR-II fluorescent agent in a pre-clinical murine model, providing a critical parameter for understanding its pharmacokinetic profile.

Principle: Renal clearance (CLR) is calculated by measuring the amount of fluorescent agent excreted in the urine over a specific interval and relating it to the plasma concentration-time profile. The most accurate method uses the area under the curve (AUC) to avoid assumptions about linear plasma decay [64].

Materials:

  • Research Reagent Solutions: See Table 1 for a complete list.
  • NIR-II contrast agent (e.g., ABY-029, IRDye 680LT, or a novel D-A-D fluorophore) [60] [36]
  • Athymic nude mice (or other relevant animal model)
  • LC-MS/MS system or validated fluorescence quantification assay [60]
  • Catheters for blood collection and intravenous injection
  • Metabolic cages for precise urine collection

Procedure:

  • Agent Administration: Administer the NIR-II agent via tail vein or retro-orbital intravenous injection at a predetermined dose (e.g., 0.0487 mg/kg for ABY-029) [60].
  • Blood Sampling: Collect serial blood samples (e.g., at 2, 5, 15, 30, 60, 120, 240, and 480 minutes post-injection) into heparinized tubes. Centrifuge samples to obtain plasma.
  • Urine Collection: House mice in metabolic cages. Collect total urine over defined time intervals (e.g., 0-4 h, 4-8 h, 8-24 h). Precisely record the volume and collection time for each interval.
  • Sample Analysis:
    • Plasma: Quantify the fluorescent agent concentration in each plasma sample using a validated method, such as the wide-field fluorescence imaging of samples in glass capillary tubes with tissue-specific calibration curves [60] or LC-MS/MS [64].
    • Urine: Dilute urine samples as necessary and quantify the agent concentration using the same analytical method.
  • Data Calculation:
    • Calculate the total amount of drug excreted in each urine collection interval (Ae).
    • Plot the plasma concentration-time data and calculate the Area Under the Curve (AUC) for the corresponding time intervals using the trapezoidal rule.
    • Calculate renal clearance (CLR) for each interval using the formula: CLR = Ae / AUC [64].
    • Report the mean CLR across intervals.

Diagram 1: Renal Clearance Determination Workflow

G Start Administer NIR-II Agent IV Blood Collect Serial Blood Samples Start->Blood Urine Collect Urine over Intervals Start->Urine AnalyzeP Analyze Plasma Concentration (Cp) Blood->AnalyzeP AnalyzeU Analyze Urine Concentration & Volume Urine->AnalyzeU CalcAUC Calculate Area Under the Curve (AUC) AnalyzeP->CalcAUC CalcAe Calculate Amount Excreted (Ae) AnalyzeU->CalcAe CalcCLR Calculate Renal Clearance CLR = Ae / AUC CalcAe->CalcCLR CalcAUC->CalcCLR

Protocol 2: In Vivo Biodistribution and Pharmacokinetic Profiling

Objective: To characterize the comprehensive PK profile and tissue biodistribution of a NIR-II contrast agent, enabling the evaluation of its circulation time and target specificity.

Principle: This protocol involves administering the agent, collecting blood for PK analysis over time, and subsequently harvesting organs to measure agent accumulation, thereby quantifying its distribution and clearance pathways [60].

Materials:

  • Research Reagent Solutions: As listed in Table 1.
  • NIR-II fluorescence imaging system (e.g., IVIS, Pearl Impulse, or custom InGaAs camera system) [31] [36]
  • Tissue homogenizer
  • Borosilicate glass capillary tubes [60]

Procedure:

  • Dosing and Blood PK: Follow steps 1-4 of Protocol 1 to establish the plasma concentration-time curve.
  • Tissue Harvesting: At predetermined terminal time points (e.g., 1 h, 24 h, 48 h post-injection), euthanize animals and harvest organs of interest (e.g., tumor, liver, spleen, kidney, muscle).
  • Tissue Processing: Homogenize entire organs or standardized tissue weights in a suitable buffer. For fluorescence-based quantification, use tissue-specific calibration curves to account for optical property differences [60].
  • Fluorescence Quantification:
    • Load homogenized tissue samples and whole blood standards into borosilicate capillary tubes.
    • Image the tubes using a wide-field fluorescence imaging system.
    • Convert mean fluorescence intensity to concentration using tissue-specific calibration curves for each fluorophore [60].
  • Data Analysis:
    • Pharmacokinetic Parameters: From plasma data, calculate total clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2) using non-compartmental analysis.
    • Biodistribution: Express tissue data as percentage of injected dose per gram of tissue (%ID/g) or as concentration relative to plasma.

Diagram 2: Key Pharmacokinetic Pathways

G Input IV Agent Input Central Central Compartment (Plasma Circulation) Input->Central Periph Peripheral Compartment (Tissue Distribution) Central->Periph Distribution Target Target Tissue (Specific Binding) Central->Target Target Specificity Clearance Elimination (Renal & Hepatic Clearance) Central->Clearance Clearance Rate Periph->Central Redistribution

Data Presentation

Table 1: Essential Research Reagent Solutions for NIR-II PK Studies

Reagent / Material Function / Application in PK Studies Key Characteristics & Examples
NIR-II Fluorophores Core imaging agent for tracking PK/BD. Small Organic Dyes (e.g., IRDye 680LT): Often show renal clearance. Targeted Agents (e.g., ABY-029): Affibody- or antibody-conjugated for specificity. D-A-D Molecules (e.g., 2FT-oCB): Engineered for bright emission >1200 nm [60] [36].
Calibration Matrix Creating tissue-specific calibration curves for accurate concentration recovery. Homogenates of relevant tissues (liver, tumor, spleen, etc.) and whole blood. Critical for accounting for tissue optical properties [60].
Borosilicate Capillaries Standardized containers for fluorescence measurement of homogenates. Provide a standardized path length for excitation light, ensuring consistent and quantitative fluorescence readings from small sample volumes [60].
Para-Aminohippuric Acid (PAH) Reference compound for measuring effective renal plasma flow. Actively secreted by renal tubules; used to quantify renal secretory function and blood flow [64].
Inulin / Iothalamate Reference marker for measuring Glomerular Filtration Rate (GFR). Inert polysaccharide filtered by glomeruli but not secreted or reabsorbed; gold standard for GFR measurement [64].

Table 2: Properties of Exemplary NIR-II Imaging Agents and Windows

Imaging Agent / Window Emission Peak (nm) Key Pharmacokinetic & Imaging Properties Primary Application Rationale
IRDye 680LT ~680 nm (NIR-I) / Can be used in NIR-II with filters Pharmacokinetically similar, untargeted control; rapid renal clearance. Paired-agent imaging (as untargeted control); baseline for vascular imaging [60].
ABY-029 ~800 nm (NIR-I/II border) EGFR-targeted Affibody molecule; pharmacokinetics influenced by targeting. Fluorescence-guided surgery; demonstrates active targeting vs. passive distribution [60].
2FT-oCB 1215 nm Engineered D-A-D molecule; ultralong molecular packing (>8 Å) inhibits π-π stacking, enhancing brightness [36]. Deep-tissue imaging with high SBR; emission matched to low-scattering windows.
SIDAG ~800 nm (NIR-I/II border) Hydrophilic derivative of ICG; shifted PK toward renal elimination; increased acute tolerance [63]. Improved tumor contrast over ICG; model for engineering hydrophilicity to modulate clearance.
NIR-IIb Window (1500-1700 nm) N/A Greatly reduced photon scattering and negligible autofluorescence. High-contrast imaging of deep structures; requires bright agents with emission in this range [31] [36].

The Scientist's Toolkit

The following reagents and tools are indispensable for conducting the experiments outlined in these protocols.

Table 3: Key Research Reagent Solutions

Category Item Specific Function
Contrast Agents IRDye 680LT (carboxylate) Untargeted, renally-cleared reference agent for PK studies [60].
ABY-029 (Anti-EGFR Affibody) Targeted agent for studying the impact of receptor binding on PK and distribution [60].
Engineered D-A-D Fluorophores (e.g., 2FT-oCB) Bright, long-wavelength emitters for high-contrast NIR-IIb imaging [36].
Analytical Tools Tissue Homogenization Buffer Medium for creating uniform tissue suspensions for quantitative fluorescence analysis.
Tissue-Specific Calibration Curves Essential standard curves for converting fluorescence signal to accurate concentration in different tissue matrices [60].
Reference Standards Para-Aminohippuric Acid (PAH) Gold standard for measuring renal plasma flow and studying active secretion [64].
Inulin or Iothalamate Gold standard for measuring Glomerular Filtration Rate (GFR) [64].

The delivery of therapeutic agents to solid tumors is a formidable challenge in oncology, primarily due to a series of biological barriers that significantly limit drug efficacy. The Enhanced Permeability and Retention (EPR) effect, first discovered by Maeda and colleagues, has long been considered the cornerstone of nanoparticle-based cancer therapy [65]. This pathophysiological phenomenon enables macromolecules and nanoparticles to selectively accumulate in tumor tissue due to leaky vasculature and defective lymphatic drainage [65] [66]. However, the journey from intravenous administration to intracellular drug release is fraught with obstacles, including rapid clearance by the Mononuclear Phagocyte System (MPS), heterogeneous EPR effects across tumor types, and poor penetration through the dense tumor microenvironment [65] [67] [68].

The clinical translation of nanomedicines has been disappointing despite promising preclinical results. With over 50,000 manuscripts published on nanoparticles for cancer by the end of 2021, only 15 antitumor nanomedicines have gained clinical approval worldwide [67]. This stark contrast highlights the critical need to understand and navigate the biological barriers that impede effective tumor targeting. This Application Note provides detailed protocols and strategies to overcome these barriers, with particular emphasis on leveraging NIR-II fluorescence imaging to monitor and validate drug delivery processes in real-time.

Understanding Key Biological Barriers

The Mononuclear Phagocyte System (MPS)

The MPS, comprising macrophages in the liver, spleen, and bone marrow, represents the first major barrier to nanoparticle delivery. Following intravenous administration, nanotherapeutics are rapidly recognized and cleared by the MPS, significantly reducing their circulation time and tumor accumulation [67] [68]. Protein corona formation – the adsorption of plasma proteins onto nanoparticle surfaces – further enhances MPS recognition and uptake [68].

The EPR Effect: Promise and Limitations

The EPR effect allows nanoparticles ranging from 40-400 nm to extravasate through the defective tumor vasculature with fenestrations of 100-780 nm and accumulate in the tumor interstitium due to impaired lymphatic drainage [65] [66]. This phenomenon can lead to drug concentrations in tumors that are 10-100 times higher than those achieved with conventional free drug administration [65].

However, the EPR effect exhibits significant heterogeneity across different tumor types, patients, and even regions within the same tumor [65] [67]. This variability stems from differences in vascular density, endothelial cell receptor expression, vascular maturation, extracellular matrix composition, tumor cell density, hypoxia, and interstitial fluid pressure [65]. Pancreatic ductal adenocarcinoma, for instance, presents particularly strong stromal barriers that limit EPR-based targeting [65].

Tumor Microenvironment Barriers

The tumor microenvironment presents multiple additional barriers including:

  • Elevated interstitial fluid pressure (IFP): Driven by fluid extravasation from leaky vessels, which creates a counter-pressure gradient that hinders nanoparticle penetration [65] [66]
  • Dense extracellular matrix (ECM): Rich in collagen and fibronectin, which creates physical barriers to nanoparticle diffusion [65] [67]
  • Aberrant tumor vasculature: Chaotic and dysfunctional blood vessels with heterogeneous perfusion [65]

Table 1: Key Biological Barriers and Their Impact on Nanodrug Delivery

Biological Barrier Main Challenges Consequences for Nanodrugs
MPS Clearance Opsonization, phagocytic uptake Short circulation half-life, reduced bioavailability
EPR Heterogeneity Inter- and intra-tumoral variability in vascular permeability Inconsistent tumor accumulation, median of only 0.7% injected dose reaches tumors [65]
Tumor Microenvironment High IFP, dense ECM, acidic pH Limited penetration and distribution, reduced cellular uptake
Cellular Uptake Endosomal entrapment, inefficient drug release Limited drug bioavailability intracellularly

The NIR-II Imaging Advantage for Tracking Tumor Targeting

Second near-infrared window (NIR-II, 1000-1700 nm) fluorescence imaging has emerged as a powerful tool for monitoring nanoparticle distribution and tumor accumulation in real-time. This technology provides significant advantages over traditional imaging modalities for validating navigation strategies across biological barriers.

Fundamental Advantages of NIR-II Imaging

NIR-II fluorescence imaging offers:

  • Deeper tissue penetration (5-20 mm) compared to visible or NIR-I imaging [58] [1]
  • Diminished photon scattering, reduced tissue autofluorescence, and higher signal-to-background ratios (SBR) [58] [1] [5]
  • Enhanced spatial resolution due to reduced light scattering at longer wavelengths [5]
  • Real-time monitoring capability of nanoparticle distribution and pharmacokinetics [58]

The improved performance in the NIR-II window stems from reduced scattering and the constructive role of moderate water absorption in suppressing multiply scattered photons that contribute to background noise [5]. This absorption preferentially depletes background signals while preserving ballistic photon signals, enhancing image contrast [5].

Advanced NIR-II Windows

Recent research has identified sub-windows within the NIR-II spectrum with optimized imaging characteristics:

  • NIR-IIa: 1300-1400 nm
  • NIR-IIb: 1500-1700 nm
  • NIR-IIx: 1400-1500 nm - provides superior image quality despite higher water absorption [5]
  • NIR-IIc: 1700-1880 nm - comparable to NIR-IIb [5]
  • NIR-III: 2080-2340 nm - proposed as the next frontier with potentially superior imaging quality [5]

Table 2: NIR-II Fluorescent Probes for Tracking Tumor Targeting

Probe Type Emission Range (nm) Advantages Limitations
Indocyanine Green (ICG) 800-1200 FDA-approved, rapid liver clearance (within 10 h) [58] Lack of tumor targeting, not activatable
Organic Cyanines 1000-1500 Tunable emission, commercial availability Compromised stability and QY with extended polymethine chain [58]
Quantum Dots (PbS/CdS) 1000-1500+ High brightness, tunable emission, narrow bandwidth [1] [5] Potential toxicity concerns
Lanthanide Nanoparticles 1000-2000 Narrow emission bands, long luminescence lifetimes, minimal photobleaching [1] Complex synthesis
D-A-D Dyes 1000-1300 High stability, large Stokes shift [1] Requires conjugation for optimal performance

Protocols for Evaluating Barrier Navigation Strategies

Protocol: NIR-II Imaging for Real-Time Tracking of Nanoparticle Pharmacokinetics

Purpose: To monitor nanoparticle circulation, tumor accumulation, and clearance in real-time using NIR-II fluorescence imaging.

Materials:

  • NIR-II fluorescent probe (e.g., ICG, PEGylated PbS/CdS QDs, or cyanine dyes)
  • Animal tumor model (subcutaneous or orthotopic)
  • NIR-II imaging system with appropriate lasers and InGaAs camera
  • Data acquisition and analysis software

Procedure:

  • Prepare fluorescent nanoparticles: Synthesize or obtain nanoparticles conjugated with NIR-II fluorophores. For QDs, use PbS/CdS core-shell structures with PEG coating for enhanced circulation [5].
  • Establish tumor model: Inoculate tumor cells (e.g., A549 or PC9 for lung cancer) subcutaneously or orthotopically in immunodeficient mice [67].
  • Administer nanoparticles: Inject nanoparticles intravenously via tail vein at appropriate concentration (e.g., 100-200 μL of 100 μM ICG equivalent).
  • Acquire time-series images: Collect NIR-II images at predetermined time points (5 min, 30 min, 2 h, 6 h, 24 h, 48 h) post-injection using consistent imaging parameters.
  • Quantify signals: Measure fluorescence intensity in tumor region and major organs (liver, spleen, kidneys) using region-of-interest analysis.
  • Calculate pharmacokinetic parameters: Determine peak tumor accumulation time, signal-to-background ratio, and clearance half-life.
  • Validate with ex vivo imaging: At endpoint, harvest organs and tumors for ex vivo imaging to confirm distribution patterns.

Notes: For optimal SBR, utilize the "off-peak" imaging method with 1400 nm long-pass detection, which can exceed the performance of strict NIR-IIb imaging [5]. The surgical window for optimal tumor resection guidance typically occurs when SBR reaches ~11.5 [58].

Protocol: Enhancing EPR Effect Using Physical and Pharmacological Methods

Purpose: To augment nanoparticle tumor accumulation by modulating tumor vasculature and microenvironment.

Materials:

  • Vasoactive agents (bradykinin, nitric oxide donors, angiotensin-converting enzyme inhibitors)
  • Physical modulation equipment (ultrasound, laser, heating apparatus)
  • NIR-II-imaging compatible nanoparticles

Procedure:

  • Characterize baseline EPR effect: Administer NIR-II nanoparticles and image tumor accumulation as described in Protocol 4.1.
  • Select enhancement strategy:
    • Pharmacological approach: Administer vasoactive agents (e.g., bradykinin) 15-30 minutes prior to nanoparticle injection [65] [66]
    • Physical approach: Apply mild hyperthermia (40-42°C), radiotherapy, or ultrasound to tumor region concurrently with or immediately before nanoparticle administration [65] [67]
  • Administer nanoparticles with EPR enhancement: Inject NIR-II nanoparticles while maintaining the enhancing stimulus.
  • Quantify enhancement: Compare tumor accumulation with and without enhancement using NIR-II imaging.
  • Optimize parameters: Titrate timing, dose, and intensity of enhancement stimuli for maximal effect.

Notes: The combination of pharmacological and physical methods often yields synergistic effects. Monitor for potential increases in systemic toxicity with vasoactive agents.

Protocol: Multiplexed NIR-II Imaging for Evaluating Heterogeneity

Purpose: To simultaneously track multiple nanoparticle formulations or target different cellular populations to assess tumor heterogeneity.

Materials:

  • Two or more NIR-II probes with non-overlapping emission spectra (e.g., QDs with emissions at 1100 nm and 1300 nm)
  • Spectral unmixing software
  • Multichannel NIR-II imaging system

Procedure:

  • Select probe combination: Choose fluorophores with emissions separated by >80 nm for effective spectral separation [1].
  • Administer probes: Inject probes simultaneously or sequentially with appropriate time intervals.
  • Acquire multichannel images: Collect signals in different emission windows (e.g., 1000-1200 nm, 1300-1500 nm) using appropriate filters.
  • Unmix signals: Use spectral unmixing algorithms to separate contributions from different probes.
  • Map distribution patterns: Generate overlays showing heterogeneous distribution of different probes within the tumor.
  • Correlate with histology: After imaging, process tumors for histological analysis to validate findings.

Notes: Lifetime-based multiplexing can complement spectral multiplexing for more complex experimental designs [1].

Research Reagent Solutions

Table 3: Essential Research Reagents for Barrier Navigation Studies

Reagent Category Specific Examples Key Functions Applications
NIR-II Fluorophores ICG, Flav7, IR-26, CH-4T [58] [1] Real-time tracking, surgical guidance Pharmacokinetic studies, tumor margin delineation
Nanoparticle Platforms PEGylated liposomes, PLGA nanoparticles, dendrimers, gold nanoparticles [65] [66] Drug encapsulation, prolonged circulation, EPR exploitation Therapeutic delivery, combination therapies
EPR Enhancers Bradykinin, nitric oxide donors, prostaglandins [65] [66] Vasodilation, increased vascular permeability Augmenting tumor accumulation
TME Modulators ECM-degrading enzymes, TGF-β inhibitors [65] [67] Reduced interstitial pressure, decreased matrix density Improving tumor penetration
Surface Modifiers PEG, CD47-derived peptides, "Self" peptides [68] Reduced opsonization, MPS evasion Prolonging circulation half-life

Strategic Framework for Barrier Navigation

The following diagram illustrates the sequential biological barriers and strategic approaches to overcome them:

G Nanoparticle Journey Through Biological Barriers cluster_0 Administration & Circulation cluster_1 Tumor Accumulation cluster_2 Penetration & Uptake cluster_3 Validation A Intravenous Injection B MPS Clearance Barrier A->B D EPR Heterogeneity Barrier C Strategies: PEGylation, Stealth Coatings Biomimetic Camouflage B->C C->D E Strategies: Vascular Modulation Physical Enhancement Multi-stage Carriers D->E F Tumor Microenvironment Barrier E->F G Strategies: Stroma-Targeting Size Reduction Enzyme Modulation F->G H NIR-II Imaging Throughout Process G->H

Integrated Approach: The CAPIR Cascade

Successful tumor targeting requires addressing the entire Circulation, Accumulation, Penetration, Internalization, and Release (CAPIR) cascade [65]. The complementary integration of passive targeting (EPR effect) with active targeting strategies is essential for improving therapeutic outcomes. This includes:

  • Pre-treatment assessment of EPR effect: Using imaging to identify patients likely to respond to nanotherapeutics [65]
  • Combination approaches: Simultaneously addressing multiple barriers rather than focusing on single obstacles [65] [68]
  • Personalized nanomedicine: Tailoring nanoparticle design and EPR enhancement strategies based on individual patient and tumor characteristics [67]

Navigating biological barriers requires a multifaceted approach that addresses the sequential challenges of MPS clearance, EPR heterogeneity, and tumor penetration. The integration of NIR-II fluorescence imaging provides an invaluable tool for developing and validating these strategies in real-time. By combining optimized nanoparticle design with EPR enhancement techniques and leveraging advanced imaging technologies, researchers can significantly improve the tumor targeting efficiency of nanotherapeutics.

The future of barrier navigation lies in personalized approaches that account for inter- and intra-tumoral heterogeneity, with treatment strategies tailored to individual patient characteristics and real-time monitoring of therapeutic response. As NIR-III imaging (2080-2340 nm) emerges, further improvements in imaging fidelity will enable even more precise evaluation of nanoparticle trafficking and tumor targeting [5].

Benchmarking NIR-II Performance: Validation in Disease Models and Comparative Analysis with Established Modalities

Within the broader thesis on Near-Infrared Window II (NIR-II, 1000–1700 nm) fluorescence imaging for deep tissue penetration research, this document provides detailed application notes and protocols for validating probe and system efficacy across the research pipeline. Compared to conventional visible (400–760 nm) and NIR-I (760–900 nm) imaging, NIR-II bioimaging offers superior spatial-temporal resolution and deeper tissue penetration (up to several centimeters) due to reduced photon scattering, low tissue autofluorescence, and minimal light absorption by biological components [33] [5]. These advantages make it an indispensable tool for fundamental and preclinical research, enabling real-time, non-invasive, and multi-dimensional investigations of in vivo biological processes [33] [4]. The following sections outline standardized protocols and key data for efficacy validation from in vitro assays through clinical patient studies.

In Vitro Validation Protocols and Data

In vitro validation establishes the foundational optical and biochemical properties of NIR-II probes, serving as a critical prerequisite for in vivo applications.

Protocol: Microarray Assay for Probe Bioconjugation and Selectivity

This protocol assesses the activity and selectivity of bioconjugates between NIR-II fluorophores and targeting ligands (e.g., antibodies, proteins) [33].

  • Key Reagents:

    • NIR-II fluorophore (e.g., IR-FGP)
    • Target protein (e.g., Serum Albumin, SA) and control protein
    • Biotin-Streptavidin system or specific ligand-receptor pairs (e.g., Erbitux (Erb) for Epidermal Growth Factor Receptor (EGFR))
    • Plasmonic fluorescence-enhancing gold slides

  • Methodology:

    • Probe Conjugation: Conjugate the NIR-II fluorophore to the targeting protein (e.g., Erbitux) per standard bioconjugation protocols.
    • Purification: Purify the conjugate using techniques like Density Gradient Ultracentrifugation (DGU) to remove unreacted dyes.
    • Microarray Printing: Spot the purified conjugates and controls onto gold slides.
    • Incubation & Washing: Incubate the microarray with target and control cell lysates (e.g., EGFR+ SCC cells and EGFR- U87MG cells), followed by stringent washing.
    • Imaging & Analysis: Image the slides using a home-built NIR-II microscope setup (e.g., 785 nm excitation, 1050 nm long-pass emission filter). Quantify the fluorescence intensity of each spot and calculate Positive/Negative (P/N) ratios to confirm selectivity [33].

Protocol: Pharmacokinetics and Biodistribution Analysis

This procedure determines the blood circulation half-life and organ-specific accumulation of NIR-II probes in small animals [33].

  • Key Reagents:

    • NIR-II fluorophore (e.g., IR-BGP6) or targeted conjugate (e.g., anti-PD-L1-BGP6)
    • Normal mice or tumor-bearing mice (e.g., MC38 model)

  • Methodology:

    • Administration: Intravenously inject the probe into the animal model.
    • Blood Kinetics: Collect blood samples at multiple time points post-injection (p.i.). Measure the fluorescence intensity in the blood to determine circulation half-life.
    • Renal Excretion: Collect urine over 24 hours and measure the accumulated fluorescence intensity to calculate the percentage of probe excreted.
    • Biodistribution: At a terminal time point (e.g., 24 h p.i.), euthanize the animal, resect major organs and tumors, and image them ex vivo using an NIR-II imaging system. Quantify the fluorescence intensity to determine the percentage of injected dose per gram of tissue (%ID/g) [33].

Table 1: Key quantitative parameters from in vitro NIR-II assays.

Parameter Experimental Result Implication
Microarray P/N Ratio Increased from 1.1 (pre-DGU) to 5.3 (post-DGU) for Erb@IR-FGP [33] DGU purification significantly improves signal-to-background ratio and probe selectivity.
Blood Half-Life ~24 minutes for IR-BGP6 [33] Indicates rapid clearance, which can be advantageous for reducing background signal.
Renal Excretion ~91% of IR-BGP6 excreted in urine within first 10 hours [33] Confirms primary clearance pathway, important for biocompatibility and toxicity assessment.
Spatial Resolution ~125 µm achieved by preclinical IR VIVO system [26] Benchmarks the high-resolution capability of NIR-II imaging systems.
Detection Sensitivity ICG concentrations as low as 30 nM (NIR-I) and 300 nM (NIR-II) [26] Highlights the sensitivity limits for fluorophore detection.

In Vivo Validation Protocols and Data

In vivo protocols translate validated probes to living systems for functional and anatomical imaging.

Protocol: Deep-Tissue Vascular and Tumor Imaging

This protocol images vascular structures and tumor margins, leveraging the deep penetration of NIR-II light [69] [5].

  • Key Reagents:

    • Bright NIR-II probe (e.g., IR-TPE Pdots, PbS/CdS Quantum Dots)
    • Tumor-bearing mouse model (e.g., BALB/c nude mice with MTCQ1 tumors)

  • Methodology:

    • Animal Preparation: Anesthetize the mouse using 2% isoflurane.
    • Probe Injection: Intravenously inject the NIR-II probe via the tail vein.
    • Image Acquisition: Place the animal in a custom-built or commercial NIR-II imaging system (e.g., equipped with a 793 nm laser for excitation and an InGaAs camera for detection). Use long-pass filters (e.g., LP1100, LP1300, LP1400) to select the desired emission window.
    • Image Analysis: Use software (e.g., MATLAB, LightField) to analyze parameters like Signal-to-Background Ratio (SBR), Signal-to-Noise Ratio (SNR), and spatial resolution [69].

Protocol: 3D Stereo Imaging of Tumor Vasculature

This protocol acquires three-dimensional information on tumor vasculature using a binocular stereo vision principle [69].

  • Key Reagents:

    • NIR-II fluorescent polymer dots (e.g., IR-TPE Pdots)
    • Tumor-bearing mouse model

  • Methodology:

    • System Setup: Use a single InGaAs camera mounted on a one-dimensional moving stage to simulate left-eye and right-eye views.
    • Image Capture: Acquire two images from slightly different angles.
    • 3D Reconstruction: Apply a stereo algorithm to match corresponding points in the two images and calculate depth information based on the disparity. The system can achieve a depth resolution of 0.6 mm and a spatial resolution of 0.15 mm [69].

Protocol: Long-Term Tracking with Transgenic Models

For studies requiring long-term observation (months), genetically encoded probes are ideal [70].

  • Key Reagents:

    • Transgenic mouse model with iRFP713 knocked into the Rosa26 locus (iRFP713flox/flox)
    • Tissue-specific Cre recombinase lines

  • Methodology:

    • Model Generation: Cross iRFP713flox/flox mice with Cre driver lines to activate iRFP713 expression in specific tissues.
    • Long-Term Imaging: Perform repeated NIR-II imaging sessions over the course of the biological process (e.g., liver regeneration over one week). The fluorescence signal is genetically encoded and passed to daughter cells, enabling continuous monitoring [70].

Table 2: Key quantitative parameters from in vivo NIR-II imaging.

Parameter Experimental Result Implication
Tissue Penetration Depth Up to ~10 mm in brain tissue using NIR-IIb/IIx windows [5] Enables visualization of deep-seated structures and tumors.
Temporal Resolution Real-time imaging capability (frames per second) [33] Allows for monitoring of dynamic processes like blood flow.
Spatial Resolution ~1.3 mm depth in mouse brain microscopy [5] Represents the deepest in vivo NIR-II fluorescence microscopy in mice brain.
Signal-to-Noise Ratio (SNR) ~5x higher in NIR-II bioluminescence vs NIR-II fluorescence [71] Elimination of excitation light leads to a dramatically cleaner signal.
Tumor-to-Normal Tissue (T/N) Ratio Up to 83.4 for ATP-responsive metastases tracing [71] Enables high-contrast detection of malignant tissue against healthy background.

Clinical Patient Validation Protocols and Data

Translating NIR-II imaging to clinical settings requires validation against current standards of care in patient studies.

Protocol: Back Table Tumor Margin Assessment

This protocol is for the ex vivo assessment of freshly excised tumor specimens to rapidly identify positive margins, a critical step in fluorescence-guided surgery (FGS) [26] [72].

  • Key Reagents:

    • Clinically available or investigational targeted tracer (e.g., cetuximab-IRDye800CW)
    • Patient tumor samples (e.g., from Penile Squamous Cell Carcinoma (PSCC) or Head and Neck Squamous Cell Carcinoma (HNSCC))

  • Methodology:

    • Tracer Administration: The targeted tracer is administered to the patient pre-operatively according to an approved clinical protocol.
    • Specimen Excision: The surgeon resects the tumor specimen.
    • Image Acquisition: Image the specimen under both NIR-I and NIR-II/SWIR clinical imaging systems (e.g., LightIR system for ambient light use, IR VIVO for enclosed imaging).
    • Data Analysis: Quantify performance using the standard Tumor-to-Background Ratio (TBR) and an adapted Contrast-to-Noise Ratio (aCNR). Use histopathological assessment of the tissue as the gold standard for validation [26] [72].

Protocol: Intraoperative Imaging Under Ambient Light

This procedure tests the feasibility of NIR-II imaging in a real-world surgical environment without the need for room blackout [26].

  • Key Reagents:

    • NIR-II fluorophore (e.g., IR-1048)
    • Clinical-grade imaging system designed for intraoperative use (e.g., LightIR)

  • Methodology:

    • System Setup: Use a compact, portable system like the LightIR, which can operate in "pulse-mode" under ambient lighting.
    • Phantom or Tissue Imaging: Image tissue-mimicking phantoms containing the NIR-II fluorophore or actual patient tissue.
    • Real-Time Subtraction: The system employs real-time background subtraction to maintain robust NIR-II contrast despite ambient light.
    • Performance Metrics: Assess depth penetration (e.g., ≥4 mm for ICG) and spatial resolution (e.g., ~250 µm) to confirm clinical utility [26].

Table 3: Key quantitative parameters from clinical NIR-II imaging studies.

Parameter Experimental Result Implication
Spatial Resolution (Clinical System) ~250 µm resolved by LightIR system [26] Sufficient resolution for identifying fine surgical margins.
Depth Penetration (Clinical) ICG detected to depths ≥4 mm [26] Allows visualization of subsurface tumors not visible to the naked eye.
Contrast-to-Noise (aCNR) Varies by cancer type; SWIR potential in PSCC, NIR better in HNSCC [72] Highlights that optimal imaging window can be cancer-specific.
Ambient Light Operation LightIR system successfully visualized subsurface targets without blackout [26] Critical for practical integration into surgical workflow.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key reagents and materials for NIR-II fluorescence imaging experiments.

Item Category Specific Examples Function & Application
NIR-II Fluorophores Organic small molecules (IR-FGP, IR-BGP6, IR-1048), Polymer dots (IR-TPE Pdots), Inorganic QDs (PbS/CdS), Rare-earth nanoparticles, Genetic encoded probes (iRFPs) [33] [70] [69] Serve as contrast agents. Choice depends on required brightness, biocompatibility, and targeting needs.
Targeting Ligands Antibodies (e.g., Cetuximab, anti-PD-L1), Peptides, Proteins Conjugated to fluorophores to enable specific binding to cellular receptors (e.g., EGFR) on target tissues [33] [72].
Imaging Systems Preclinical (IR VIVO), Clinical/Intraoperative (LightIR), Custom-built setups with InGaAs cameras [26] [69] Detect NIR-II fluorescence. Systems vary in portability, sensitivity, and suitability for in vivo vs. clinical use.
Key Optical Components Laser Diodes (e.g., 793 nm, 808 nm), Long-Pass Filters (e.g., LP1100, LP1250, LP1400), InGaAs Cameras [26] [69] Components for excitation (lasers), emission filtering (to block excitation light), and detection (cameras).
Animal Models Wild-type mice, Transgenic models (e.g., iRFP713flox/flox), Tumor-bearing models (e.g., MC38, MTCQ1) [33] [70] [69] Provide the in vivo context for evaluating probe performance and imaging biological processes.

Experimental Workflow and System Comparison Diagrams

NIR-II Bioimaging Validation Workflow

The following diagram outlines the core logical pathway for validating NIR-II imaging efficacy from basic research to clinical application.

G Start Start: NIR-II Probe/System Development InVitro In Vitro Validation Start->InVitro OptChar Optical Characterization (Quantum Yield, Brightness) InVitro->OptChar BioAssay Bioconjugation & Selectivity Assays (e.g., Microarray) InVitro->BioAssay PK Pharmacokinetics Analysis (Blood Half-Life, Excretion) InVitro->PK InVivo In Vivo Validation OptChar->InVivo BioAssay->InVivo PK->InVivo Vascular Vascular & Anatomical Imaging InVivo->Vascular TumorImg Tumor Targeting & Margin Delineation InVivo->TumorImg LongTerm Long-Term Tracking (using transgenic models) InVivo->LongTerm Clinical Clinical Patient Validation Vascular->Clinical TumorImg->Clinical LongTerm->Clinical ExVivo Ex Vivo Specimen Imaging (Back Table Margin Assessment) Clinical->ExVivo Intraop Intraoperative Imaging (Under Ambient Light) Clinical->Intraop Compare Compare vs. Standard of Care (NIR-I & Histopathology) Clinical->Compare Impact Outcome: Improved Surgical Precision and Patient Outcomes ExVivo->Impact Intraop->Impact Compare->Impact

NIR-II vs. NIR-I Imaging Performance Comparison

This diagram visually compares the key performance metrics between NIR-I and NIR-II imaging windows, based on data from preclinical and clinical studies.

G Title NIR-I vs. NIR-II Imaging Performance NIRI NIR-I Imaging (700-900 nm) Scattering Photon Scattering NIRI->Scattering -> Autofluor Tissue Autofluorescence NIRI->Autofluor -> Penetration Tissue Penetration Depth NIRI->Penetration -> Resolution Spatial Resolution NIRI->Resolution -> Contrast Image Contrast (SNR/TBR) NIRI->Contrast -> NIRII NIR-II Imaging (1000-1700 nm) NIRII->Scattering -> NIRII->Autofluor -> NIRII->Penetration -> NIRII->Resolution -> NIRII->Contrast -> NIRHI High Scattering->NIRHI NIRIIHI Low Scattering->NIRIIHI NIRLO High Autofluor->NIRLO NIRIILO Low Autofluor->NIRIILO NIRMed Medium Penetration->NIRMed NIRIIMed High Penetration->NIRIIMed Resolution->NIRMed Resolution->NIRIIMed Contrast->NIRMed Contrast->NIRIIMed

Within the broader thesis on NIR-II fluorescence imaging for deep-tissue penetration research, this document establishes detailed application notes and protocols for the quantitative assessment of multi-organ dysfunction and therapeutic response. Traditional methods for monitoring organ function, such as pathological analysis and the measurement of biomarkers like creatinine, are often invasive, slow, and lack the specificity for early detection of dysfunction [73] [74]. Consequently, there is a pressing need for non-invasive, real-time technologies that enable the precise longitudinal monitoring of multiple organs simultaneously.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) has emerged as a powerful solution to this challenge. Due to reduced light scattering, absorption, and negligible tissue autofluorescence in this spectral region, NIR-II imaging offers superior tissue penetration depth (up to several centimeters) and a high spatiotemporal resolution compared to imaging in the visible or first near-infrared (NIR-I) windows [52] [75] [76]. These advantages make it an ideal modality for the real-time, quantitative assessment of physiological processes and drug-induced pathologies across deep tissues [77]. This protocol details the application of a multiplexed NIR-II fluorescent bioimaging method for the simultaneous evaluation of liver, kidney, and gastrointestinal function, providing a novel approach for enhancing the evaluation of drug side effects and therapeutic interventions [73].

Key Advantages of NIR-II Imaging for Quantitative Assessment

The quantitative assessment of multi-organ dysfunction requires high-fidelity data, which is enabled by the unique physical properties of the NIR-II window.

  • Deep Tissue Penetration and High Resolution: The reduced scattering of light in the NIR-II region allows photons to travel deeper into biological tissues and maintain a straighter path. This results in imaging penetration depths of up to ~1 cm and provides high spatial resolution, which is crucial for resolving the fine structures of deep-seated organs [52] [76].
  • High Signal-to-Noise Ratio (SNR): Biological tissues exhibit significant autofluorescence in the visible and NIR-I regions. In contrast, autofluorescence is minimal in the NIR-II window, leading to a dramatically improved signal-to-background ratio [52] [77]. A high SNR is a prerequisite for precise quantification, as it allows for the clear distinction of the probe signal from background noise.
  • Real-Time and Dynamic Monitoring: Fluorescence imaging provides a non-invasive means to monitor biological processes in real-time. This capability allows for the dynamic tracking of probe metabolism and clearance in organs, enabling the quantitative assessment of organ function over time without the need to sacrifice animal cohorts at different time points [73] [75].

The following table summarizes the quantitative benefits of NIR-II imaging compared to traditional optical windows.

Table 1: Comparative Advantages of NIR-II Imaging for Quantitative Assessment

Characteristic Visible/NIR-I Imaging NIR-II Imaging Impact on Quantitative Assessment
Tissue Penetration Limited (1-3 mm) [76] Deep (~1 cm) [52] Enables imaging of deep organs like liver and kidneys.
Spatial Resolution Lower (blurred by scattering) Higher (< 10 µm through skull) [77] Allows precise localization of signal to specific organ structures.
Signal-to-Noise Ratio Lower due to autofluorescence Significantly higher [52] [77] Improves accuracy of signal quantification and detection sensitivity.
Tissue Scattering High Reduced (decreases with λ^α) [52] Minimizes image distortion, leading to more accurate measurements.

The Scientist's Toolkit: Research Reagent Solutions

The successful implementation of this protocol relies on a suite of specialized reagents and equipment. The core components are NIR-II fluorescent probes, which can be broadly categorized into inorganic and organic types, each with distinct properties suitable for different applications.

Table 2: Essential Research Reagents and Materials for NIR-II Imaging

Item Category Specific Examples Function and Key Characteristics
NIR-II Fluorophores Hemicyanine Dyes (HDs) [73], Organic Small Molecules (CH1055, Y6CT) [75] [78], Lanthanide-Doped Nanoparticles (DCNPs) [76], Quantum Dots (Agâ‚‚S QDs) [76] Function as contrast agents. HDs and small molecules offer synthetic tunability and better biocompatibility; inorganic probes like DCNPs and QDs offer high photostability and quantum yield.
Targeting Moieties Antibodies, Peptides, Ligands Conjugated to fluorophores to enable specific binding to cellular biomarkers or organ structures, enhancing localization and signal specificity.
Molecular Scaffolds A-D-A type conjugated molecules (e.g., Y6CT) [78], Donor-Acceptor-Donor (D-A-D) structures [75] Form the core of organic NIR-II fluorophores, allowing for precise tuning of absorption/emission wavelengths and brightness through chemical synthesis.
Nanoparticle Matrix DSPE-mPEG2000 [78] Used to encapsulate hydrophobic organic dyes into nanoparticles (e.g., Y6CT-NPs), improving aqueous solubility, biocompatibility, and circulation time.
Activation Probes Activatable Probes (e.g., DPM-HD3-CO) [79] "Turn-on" in response to specific biomarkers (e.g., carbon monoxide), providing high specificity for detecting pathological events.
Imaging Instrumentation InGaAs Camera [52] [75] Essential detector for capturing light in the 900-1700 nm range. Requires cooling to reduce thermal noise for high-sensitivity NIR-II imaging.
Excitation Sources Lasers (808 nm), White-light sources (Laparoscopic LED) [78] Activate the NIR-II fluorophores. Lasers are common, but clinical white-light sources offer homogeneous illumination and safety for translation.

Experimental Protocols

This section provides a detailed, step-by-step methodology for applying multiplexed NIR-II imaging to assess drug-induced multi-organ dysfunction, using cisplatin and aristolochic acids as model compounds [73].

Protocol 1: Multiplexed In Vivo NIR-II Imaging of Organ Dysfunction

Objective: To simultaneously monitor the real-time dysfunction of the liver, kidneys, and gastrointestinal tract in a live animal model following drug administration.

Materials:

  • Animal model (e.g., mouse)
  • Synthesized NIR-II hemicyanine dyes (HDs) with distinct emissions [73]
  • Drug of interest (e.g., Cisplatin, Aristolochic Acid-I/II)
  • NIR-II Imaging System equipped with an InGaAs camera
  • Anesthesia system (e.g., isoflurane vaporizer)
  • Heating pad for maintaining body temperature
  • Physiological saline

Procedure:

  • Animal Model Preparation: Induce multi-organ dysfunction in the animal model. For example, administer a single intraperitoneal injection of cisplatin (e.g., 15-20 mg/kg) to induce liver, kidney, and gastric injuries. Allow the pathology to develop for 2-3 days.
  • Dye Preparation: Prepare fresh solutions of the selected NIR-II HDs in sterile physiological saline or PBS. Filter-sterilize the solution using a 0.22 µm filter.
  • Dye Administration: Intravenously inject the cocktail of NIR-II HDs via the tail vein. A typical injection volume for a mouse is 100-200 µL, with the dye concentration optimized for a strong signal (e.g., absorbance ~0.1-1 at the excitation wavelength).
  • Image Acquisition: a. Anesthetize the animal and place it in a prone or supine position on the imaging stage. b. Set the imaging parameters: excitation laser power (e.g., 808 nm, 100 mW/cm²), exposure time (e.g., 50-200 ms), and field of view. c. Acquire time-series images over a period of 60-120 minutes post-injection. Ensure consistent imaging parameters throughout the experiment. d. For multiplexed imaging, use optical filters to separate the signals from different HDs based on their emission wavelengths.
  • Data Analysis: a. Draw regions of interest (ROIs) around the liver, kidneys, stomach, and intestines. b. Plot the fluorescence intensity versus time for each organ to generate time-activity curves. c. Quantify key pharmacokinetic parameters: * Time-to-Peak (TTP): The time taken for the signal in each organ to reach its maximum intensity. * Half-Life (t₁/â‚‚): The time for the signal intensity to reduce by half from its peak, indicating clearance rate. * Area Under the Curve (AUC): The integral of the time-activity curve, representing total dye exposure.

Validation: Correlate the in vivo imaging findings with gold-standard ex vivo analyses, such as histopathological examination (H&E staining) and transcriptomics of the extracted organs [73].

Protocol 2: Quantitative Assessment of Therapeutic Response

Objective: To non-invasively monitor the response of a diseased organ to a therapeutic intervention using NIR-II imaging.

Materials:

  • Animal model with induced condition (e.g., hepatic ischemia-reperfusion [78] or tumor)
  • NIR-II imaging agent (e.g., Y6CT-NPs [78] or a targeted NIR-II probe)
  • Therapeutic compound
  • NIR-II Imaging System

Procedure:

  • Baseline Imaging: Prior to therapy, administer the NIR-II probe and acquire baseline images as described in Protocol 1.
  • Therapeutic Intervention: Administer the therapeutic agent.
  • Longitudinal Monitoring: At predetermined time points (e.g., days 1, 3, 7 post-therapy), re-administer the NIR-II probe and repeat the imaging process. Keep all imaging parameters identical to the baseline session.
  • Data Analysis: a. Quantify the fluorescence signal in the target organ (e.g., injured liver region or tumor) at each time point. b. Normalize the post-treatment signals to the baseline signal. c. Plot the normalized signal intensity over time. A decreasing trend indicates a positive therapeutic response (e.g., reduction in inflammation or tumor size), while a stable or increasing signal may indicate non-responsiveness or disease progression.

Data Presentation and Analysis

The quantitative data derived from NIR-II imaging experiments should be systematically organized to facilitate comparison and interpretation. The following table provides a template for summarizing key pharmacokinetic parameters from a multi-organ dysfunction study.

Table 3: Quantitative Pharmacokinetic Parameters from NIR-II Imaging of Organ Function

Organ Experimental Group Time-to-Peak (TTP, min) Signal Half-Life (t₁/₂, min) AUC (a.u.) Interpretation
Liver Control 5.2 ± 0.8 45.3 ± 5.1 10500 ± 850 Normal uptake and metabolism
Cisplatin-Treated 8.7 ± 1.1 92.5 ± 9.8 21800 ± 1200 Impaired metabolic function
Kidney Control 3.5 ± 0.5 25.1 ± 3.2 5800 ± 450 Normal filtration and clearance
Cisplatin-Treated 6.8 ± 0.9 65.4 ± 7.2 15200 ± 1100 Acute kidney injury
Stomach Control - - - Low background signal
AA-I-Treated 15.2 ± 2.5 >120 9500 ± 800 Gastric emptying disorder [73]

Workflow and Signaling Visualizations

The following diagrams illustrate the core experimental workflow and molecular design principles underlying the NIR-II imaging protocol.

G Start 1. Animal Model Preparation (Induce dysfunction) A 2. Administer NIR-II Dye Cocktail (e.g., Hemicyanine Dyes) Start->A B 3. In Vivo NIR-II Image Acquisition (Time-series over 60-120 min) A->B C 4. Quantitative Image Analysis (ROI-based intensity tracking) B->C D 5. Pharmacokinetic Modeling (TTP, t½, AUC calculation) C->D E 6. Validation (Histopathology, Transcriptomics) D->E End Data Interpretation & Conclusion E->End

Diagram 1: Experimental Workflow for NIR-II Organ Dysfunction Assessment

G Light White-light/Laser Excitation Interaction Light-Tissue Interaction Light->Interaction Probe NIR-II Imaging Probe Probe->Interaction Emission NIR-II Emission (1000-1700 nm) Interaction->Emission Low Scattering Low Absorption Detection Signal Detection by InGaAs Camera Emission->Detection Output Quantitative Data (High SNR, Deep Penetration) Detection->Output

Diagram 2: NIR-II Imaging Principle and Advantage

Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) has emerged as a transformative modality for in vivo biological imaging, addressing critical limitations of both conventional clinical imaging systems and earlier optical techniques. This application note provides a structured comparison of NIR-II fluorescence imaging against established clinical modalities (MRI, CT, PET) and first near-infrared window (NIR-I) fluorescence imaging, with emphasis on their respective capabilities for deep-tissue imaging research. The content is framed within the broader thesis that NIR-II imaging offers superior performance for specific preclinical and potential clinical applications requiring high spatial resolution, deep penetration, and real-time visualization capabilities.

The fundamental advantage of NIR-II imaging stems from reduced photon scattering and minimal tissue autofluorescence at longer wavelengths, which collectively enhance penetration depth and improve signal-to-background ratios (SBR). Recent research has further refined our understanding of optimal sub-windows within the NIR-II spectrum, including regions with higher water absorption (1400-1500 nm and 1880-2080 nm) that paradoxically improve imaging contrast by preferentially attenuating scattered background photons [24]. For researchers and drug development professionals, understanding these comparative advantages is crucial for selecting appropriate imaging methodologies for specific experimental and diagnostic challenges.

Technical Comparison of Imaging Modalities

Performance Metrics and Characteristics

Table 1: Head-to-Head Comparison of Key Imaging Modalities

Parameter NIR-II Fluorescence NIR-I Fluorescence MRI CT PET
Spatial Resolution ∼µm range (∼25-50 µm in vivo) [32] ∼µm range (limited by scattering) [32] ∼µm (preclinical) to mm (clinical) [80] ∼µm (preclinical) to mm (clinical) [80] 1-2 mm (preclinical) to 5-7 mm (clinical) [80]
Penetration Depth Up to several centimeters [32] [34] <1 cm [32] Unlimited Unlimited Unlimited
Temporal Resolution Excellent (seconds to minutes) [80] Excellent (seconds to minutes) [80] Minutes to hours [32] Excellent (seconds) [32] Minutes to hours [32]
Sensitivity High (nM-pM) [80] High (nM-pM) [80] Low (µM-mM) [80] Low High (pM-fM) [80]
Ionizing Radiation No [32] No [32] No Yes [32] Yes [32]
Quantitative Capability Moderate Moderate Excellent (functional) Excellent (anatomical) Excellent (metabolic)
Molecular Specificity Excellent [32] Excellent [32] Good with targeted agents Poor Excellent [32]
Real-time Imaging Yes [34] Yes [34] No Yes No
Cost & Accessibility Moderate Low High [32] Moderate [32] High [32]
Contrast Agent Requirements Required (fluorophores) [32] Required (fluorophores) [32] Optional (gadolinium, iron oxide) Iodine-based required Radiolabeled tracers required

Table 2: NIR-II Sub-window Characteristics for Deep-Tissue Imaging

Spectral Window Wavelength Range (nm) Key Characteristics Optimal Applications
NIR-IIa 1300-1400 [24] Reduced scattering, low water absorption Deep-tissue vascular imaging [24]
NIR-IIx 1400-1500 [24] Moderate water absorption improves contrast High-contrast imaging in aqueous tissues [24]
NIR-IIb 1500-1700 [24] Further reduced scattering Superficial high-resolution imaging
NIR-IIc 1700-1880 [24] Low scattering, emerging applications Imaging in adipose-rich environments [24]
Extended NIR-II 1880-2080 [24] High water absorption for maximal contrast Ultra-high contrast for complex backgrounds [24]

Comparative Analysis of Strengths and Limitations

NIR-II Fluorescence Imaging excels in high-resolution real-time visualization of molecular targets with exceptional sensitivity. The technique leverages reduced scattering of longer wavelengths, enabling deeper penetration (up to several centimeters) compared to NIR-I while maintaining micron-scale resolution [32] [34]. Recent evidence demonstrates that previously avoided spectral regions near water absorption peaks (∼1450 nm and ∼1930 nm) actually provide superior contrast by preferentially attenuating longer-path scattered photons [24]. This paradigm shift enables high-contrast imaging in the 1880-2080 nm window when sufficiently bright fluorophores are employed.

Clinical Modalities (MRI, CT, PET) provide whole-body capabilities with complementary strengths. MRI offers excellent soft-tissue contrast without radiation exposure but suffers from low sensitivity and slow imaging times [32]. CT provides exceptional bone visualization and rapid acquisition but involves ionizing radiation and poor soft-tissue contrast [80]. PET delivers unparalleled sensitivity for metabolic processes but requires radioactive tracers and offers limited spatial resolution [80] [32].

NIR-I Fluorescence Imaging (700-900 nm) maintains advantages in instrument availability and established dye chemistry but is fundamentally limited by significant tissue scattering and autofluorescence, constraining penetration depth to <1 cm with compromised resolution in deep tissues [32].

Experimental Protocols for NIR-II Fluorescence Imaging

Protocol 1: High-Contrast Vascular Imaging in the 1880-2080 nm Window

Principle: Exploits the high water absorption around 1930 nm to preferentially attenuate scattered photons, dramatically improving signal-to-background ratio in vascular imaging [24].

Materials:

  • PbS/CdS core-shell quantum dots (emission peak ∼1700-2000 nm) [24]
  • NIR-II imaging system with InGaAs camera sensitive to 2000+ nm
  • 1064 nm laser source (200 mW/cm²)
  • Animal immobilization platform
  • Heating pad for physiological maintenance

Procedure:

  • Synthesize water-soluble PbS/CdS QDs per established protocols with polyethylene glycol (PEG) coating [24].
  • Administer QDs intravenously via tail vein (200 µL of 100 nM concentration in saline).
  • Anesthetize animal and position for imaging region of interest.
  • Set imaging system to 1880-2080 nm spectral window using appropriate longpass filters.
  • Apply 1064 nm excitation at 200 mW/cm² power density.
  • Acquire time-series images at 100-500 ms exposure for 20 minutes post-injection.
  • Process images using background subtraction and contrast optimization algorithms.

Key Considerations: The bright fluorescence of PbS/CdS QDs must overcome significant water absorption attenuation. Optimal results require emission matching the 1880-2080 nm window [24].

Protocol 2: Multimodal NIR-II Fluorescence Imaging with Protein-Seeking Dyes

Principle: Creates biomimetic NIR-II fluorescent proteins through covalent binding of protein-seeking dyes to serum albumin, enhancing brightness and photostability for prolonged imaging sessions [8].

Materials:

  • CO-1080 dye or similar protein-seeking NIR-II fluorophore
  • Human serum albumin (HSA)
  • Reaction buffer (pH 7.4)
  • Ultrafiltration concentrators (50 kDa MWCO)
  • InGaAs camera system

Procedure:

  • Prepare CO-1080 dye solution in DMSO (10 mM stock).
  • Mix HSA (10 µM) with CO-1080 (10 µM) in reaction buffer.
  • Incubate at 60°C for 2 hours to facilitate covalent binding.
  • Purify HSA@CO-1080 complex using ultrafiltration (50 kDa MWCO).
  • Characterize optical properties: absorption at 1044 nm, emission at 1079 nm.
  • Administer HSA@CO-1080 intravenously (150 µL of 50 µM concentration).
  • Image with 1064 nm excitation, collecting emission in 1100-1300 nm window.
  • For multicolor imaging, combine with NIR-I fluorophores excited at 808 nm.

Key Considerations: The hydrophobic cavity of HSA restricts molecular motion of covalently bound dyes, reducing non-radiative decay and enhancing fluorescence intensity up to 22-fold [8].

Visualization of Experimental Workflows

NIR-II Fluorophore Design and Performance Optimization

G Molecular Engineering Molecular Engineering D-A-D Framework D-A-D Framework Molecular Engineering->D-A-D Framework Cyanine Derivatives Cyanine Derivatives Molecular Engineering->Cyanine Derivatives BODIPY Derivatives BODIPY Derivatives Molecular Engineering->BODIPY Derivatives Xanthene Dyes Xanthene Dyes Molecular Engineering->Xanthene Dyes Brightness Enhancement Brightness Enhancement D-A-D Framework->Brightness Enhancement Cyanine Derivatives->Brightness Enhancement BODIPY Derivatives->Brightness Enhancement Xanthene Dyes->Brightness Enhancement Intramolecular Locking Intramolecular Locking Brightness Enhancement->Intramolecular Locking Steric Shielding Steric Shielding Brightness Enhancement->Steric Shielding TICT Suppression TICT Suppression Brightness Enhancement->TICT Suppression Aggregation-Induced Emission Aggregation-Induced Emission Brightness Enhancement->Aggregation-Induced Emission Enhanced NIR-II Performance Enhanced NIR-II Performance Intramolecular Locking->Enhanced NIR-II Performance Steric Shielding->Enhanced NIR-II Performance TICT Suppression->Enhanced NIR-II Performance Aggregation-Induced Emission->Enhanced NIR-II Performance

Figure 1: Strategic approaches for developing high-performance NIR-II fluorophores through molecular engineering and brightness enhancement techniques [81] [32].

Mechanism of High-Contrast Imaging in Water Absorption Windows

G Photon-Tissue Interaction Photon-Tissue Interaction Ballistic Photons (Short Path) Ballistic Photons (Short Path) Photon-Tissue Interaction->Ballistic Photons (Short Path) Scattered Photons (Long Path) Scattered Photons (Long Path) Photon-Tissue Interaction->Scattered Photons (Long Path) Minimal Attenuation Minimal Attenuation Ballistic Photons (Short Path)->Minimal Attenuation Preferential Attenuation Preferential Attenuation Scattered Photons (Long Path)->Preferential Attenuation Water Absorption Water Absorption Water Absorption->Minimal Attenuation Water Absorption->Preferential Attenuation High Signal Retention High Signal Retention Minimal Attenuation->High Signal Retention Background Suppression Background Suppression Preferential Attenuation->Background Suppression Enhanced Image Contrast Enhanced Image Contrast High Signal Retention->Enhanced Image Contrast Background Suppression->Enhanced Image Contrast

Figure 2: Mechanism demonstrating how water absorption in specific NIR-II windows (1400-1500 nm and 1880-2080 nm) preferentially attenuates scattered background photons, enhancing image contrast [24].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagents for NIR-II Fluorescence Imaging

Reagent Category Specific Examples Function & Characteristics Application Context
Inorganic Nanoparticles PbS/CdS QDs [24], Agâ‚‚S QDs [82] Bright, photostable emitters with tunable NIR-II emission Deep-tissue imaging (1880-2080 nm window) [24]
Organic Small Molecules D-A-D fluorophores [32], Cyanines [32] Well-defined structures, favorable biocompatibility Molecular-targeted imaging, rapid clearance studies
Biomimetic Systems HSA@CO-1080 [8], Protein-seeking dyes Enhanced brightness via restricted molecular motion Long-circulating vascular imaging, lymphography
Surface Modification Agents PEG derivatives, targeting peptides Improve biocompatibility and target specificity Tumor-specific imaging, pharmacokinetic optimization
Detection Systems InGaAs cameras [34], Spectral filters Capture NIR-II photons with high sensitivity System setup for various NIR-II sub-windows

NIR-II fluorescence imaging represents a powerful modality that addresses fundamental limitations of both conventional clinical imaging and earlier optical techniques. Its unique combination of high spatial resolution, deep-tissue penetration, and real-time imaging capability positions it as an invaluable tool for preclinical research and emerging clinical applications.

The strategic selection of NIR-II sub-windows, particularly those leveraging water absorption characteristics (1400-1500 nm and 1880-2080 nm), enables unprecedented contrast for visualizing complex biological structures and processes. Continued development of brighter fluorophores through molecular engineering strategies like intramolecular covalent bond locking [81] and biomimetic protein-dye complexes [8] will further expand the capabilities of NIR-II imaging.

For researchers and drug development professionals, integrating NIR-II fluorescence imaging with established modalities like MRI and PET offers a multimodal approach that leverages the respective strengths of each technology. This synergistic strategy promises to accelerate both fundamental biological discovery and the translation of novel diagnostic and therapeutic agents.

Fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) represents a paradigm shift in biomedical imaging, offering unprecedented capabilities for deep-tissue visualization with high spatial resolution. This technology leverages the favorable optical properties of biological tissues in this spectral range, including reduced photon scattering, lower tissue autofluorescence, and minimized light absorption by key tissue components such as hemoglobin and water [31] [14]. These intrinsic advantages enable NIR-II fluorescence imaging to achieve millimeter-to-centimeter penetration depths with micron-scale resolution, far surpassing the capabilities of traditional NIR-I imaging (700-900 nm) [31] [58]. The burgeoning applications span from intraoperative surgical guidance and tumor margin delineation to vascular imaging and therapeutic monitoring, positioning NIR-II technology at the forefront of next-generation diagnostic and therapeutic platforms [26] [58].

Despite these remarkable advantages, the clinical translation of NIR-II imaging agents faces significant challenges centered on biocompatibility, toxicity, and regulatory compliance. The limited pool of effective NIR-II fluorophores, coupled with concerns regarding potential long-term accumulation and immunogenic responses, presents substantial barriers to clinical adoption [26] [34]. Many promising NIR-II probes exhibit suboptimal pharmacokinetics, inadequate target-to-background ratios, and insufficient brightness in the longer-wavelength NIR-II sub-windows (NIR-IIa, 1300-1400 nm; NIR-IIb, 1500-1700 nm) where imaging performance is optimal [14] [36]. Furthermore, the high cost of specialized imaging equipment, particularly InGaAs cameras required for NIR-II detection, has limited accessibility and widespread clinical application [31] [26]. This application note provides a comprehensive framework for addressing these translational challenges, with structured protocols for evaluating the safety and efficacy of NIR-II contrast agents within a regulatory context.

Biocompatibility and Toxicity Profiles of NIR-II Imaging Agents

The biological interactions of NIR-II imaging agents vary significantly based on their material composition, surface chemistry, and structural characteristics. A thorough understanding of these properties is essential for rational probe design and clinical development.

Classification and Properties of NIR-II Fluorophores

Table 1: Comparative Analysis of NIR-II Fluorophore Classes and Their Properties

Fluorophore Class Examples Key Advantages Biocompatibility Concerns Current Status
Organic Small Molecules ICG, Cyanine dyes, BODIPY, Donor-Acceptor-Donor (D-A-D) dyes Tunable pharmacokinetics, renal clearance, well-defined chemical structures [58] [83] Moderate quantum yield, potential photobleaching, variable stability in biological environments [14] [58] ICG FDA-approved for NIR-I; NIR-II analogs in preclinical development [58]
Inorganic Nanomaterials Quantum Dots (QDs), Rare-Earth-Doped Nanoparticles (RENPs), Single-Walled Carbon Nanotubes (SWCNTs) High photostability, strong fluorescence intensity, tunable optical properties [31] [34] Long-term accumulation potential, metal ion leakage, undefined clearance pathways [26] [34] Primarily preclinical; significant toxicity concerns require resolution
Hybrid Materials Dye-loaded nanoparticles, polymer-fluorophore conjugates Enhanced brightness, multimodal capabilities, tailored pharmacokinetics [34] Complex manufacturing, batch-to-batch variability, uncertain regulatory pathway Early-stage development with promising preclinical results

Critical Quality Attributes (CQAs) for Biocompatibility Assessment

The biological safety of NIR-II imaging agents is governed by a set of Critical Quality Attributes (CQAs) that must be carefully evaluated:

  • Size and Surface Characteristics: Nanoparticle size directly influences biodistribution, cellular uptake, and clearance pathways. Materials smaller than 5-6 nm typically undergo renal clearance, while larger particles may accumulate in the reticuloendothelial system (liver and spleen) [14]. Surface charge (zeta potential) affects protein corona formation and subsequent immune recognition, with highly positive or negative surfaces generally exhibiting increased cytotoxicity [34].

  • Chemical Stability and Degradation Products: Fluorophores must demonstrate stability in physiological environments to prevent release of potentially toxic components. For inorganic nanomaterials, this includes assessment of metal ion leaching under acidic conditions (e.g., lysosomal environments) [34]. Degradation pathways should be clearly elucidated, with toxicological profiling of all major degradation products.

  • Photophysical Properties and Light-Tissue Interactions: High quantum yield enables lower dosing for effective imaging, directly reducing potential toxicity [14]. Additionally, photostability minimizes generation of reactive oxygen species (ROS) that could cause tissue damage, particularly important for applications requiring prolonged illumination [36].

Experimental Protocols for Safety and Efficacy Assessment

Robust assessment protocols are essential for generating the comprehensive safety and efficacy data required for regulatory submissions.

Protocol 1: In Vitro Biocompatibility Screening

Objective: To evaluate baseline cytotoxicity, cellular uptake, and inflammatory responses induced by NIR-II imaging agents.

Materials:

  • Test article: NIR-II fluorophore (various concentrations)
  • Cell lines: Primary macrophages (e.g., THP-1), hepatocytes, and renal proximal tubule epithelial cells
  • Assay kits: MTT/XTT for viability, LDH for membrane integrity, ELISA for cytokine profiling (IL-1β, IL-6, TNF-α)
  • Imaging equipment: Confocal microscope with NIR-II detection capabilities

Methodology:

  • Dose-Ranging Studies: Expose cells to fluorophore concentrations spanning three orders of magnitude (0.1-100 μg/mL) for 4, 24, and 72 hours to identify toxicity thresholds [34].
  • Viability Assessment: Quantify metabolic activity (MTT assay) and membrane integrity (LDH release) following exposure, with ≥80% viability relative to untreated controls considered acceptable [34].
  • Inflammatory Response Profiling: Measure secretion of pro-inflammatory cytokines in macrophage cultures after 24-hour exposure using ELISA.
  • Cellular Uptake and Localization: Incubate cells with fluorophores for 4 hours, wash thoroughly, and visualize internalization using NIR-II microscopy. Quantify uptake efficiency via fluorescence-activated cell sorting (FACS).
  • Reactive Oxygen Species (ROS) Detection: Employ DCFH-DA assay to measure fluorophore-induced ROS generation, with and without light irradiation.

Data Analysis: Calculate IC50 values for cytotoxicity and no-observed-adverse-effect-level (NOAEL) concentrations. Correlation analysis between physicochemical properties (size, charge) and biological responses guides material optimization.

Protocol 2: In Vivo Toxicity and Pharmacokinetics

Objective: To assess systemic toxicity, organ accumulation, and clearance pathways in relevant animal models.

Materials:

  • Animal model: Rodents (mice/rats) and optionally higher species (e.g., rabbits)
  • NIR-II fluorophore at proposed clinical formulation
  • Imaging system: NIR-II fluorescence imager (e.g., IR-VIVO or LightIR systems) [26]
  • Histopathology equipment and clinical chemistry analyzers

Methodology:

  • Dose Escalation Study: Administer single doses of fluorophore at 1x, 5x, and 10x the anticipated clinical imaging dose via intended route (typically intravenous) [58].
  • Acute Toxicity Monitoring: Record clinical observations, body weight, and food consumption daily for 14 days. Euthanize subsets at 24 hours, 7 days, and 14 days for comprehensive gross necropsy.
  • Pharmacokinetic Profiling: Collect serial blood samples at predetermined timepoints (5, 15, 30 min; 1, 2, 4, 8, 24, 48 h) post-administration. Quantify blood circulation half-life (α and β phases) using fluorescence measurements [14].
  • Biodistribution Analysis: At terminal timepoints, harvest major organs (heart, liver, spleen, lungs, kidneys, brain) for ex vivo NIR-II imaging and quantitative fluorescence analysis. Calculate percentage injected dose per gram of tissue (%ID/g) [58].
  • Clinical Pathology: Perform comprehensive hematology and clinical chemistry panels at 24 hours and 14 days post-administration. Compare treated groups with vehicle controls.
  • Histopathological Examination: Process organs for H&E staining and specialized staining as needed. Evaluate for treatment-related lesions by a board-certified veterinary pathologist blinded to treatment groups.

Data Analysis: Establish maximum tolerated dose (MTD) and no-observed-adverse-effect-level (NOAEL). Calculate key pharmacokinetic parameters: Cmax, Tmax, AUC, clearance rate, and volume of distribution.

Protocol 3: Efficacy Assessment in Disease Models

Objective: To validate imaging efficacy and target engagement in physiologically relevant models.

Methodology:

  • Disease Model Establishment: Utilize orthotopic or transgenic models that recapitulate human disease pathology (e.g., glioblastoma, metastatic cancer) [34].
  • Imaging Protocol Optimization: Determine optimal imaging timepoints based on pharmacokinetic data, typically corresponding to peak target-to-background ratios [58].
  • Quantitative Performance Metrics: Calculate signal-to-background ratios (SBR) and contrast-to-noise ratios (CNR) at the target site versus adjacent normal tissue [26] [36].
  • Correlation with Histology: After terminal imaging, excise tissues for immunohistochemical validation of target expression and spatial correlation with fluorescence signals.

G Start NIR-II Probe Development PC Physicochemical Characterization Start->PC InVitro In Vitro Biocompatibility PC->InVitro AnimalModels Animal Model Evaluation InVitro->AnimalModels Tox Comprehensive Toxicology AnimalModels->Tox PK Pharmacokinetics & Metabolism AnimalModels->PK Eff Efficacy Studies AnimalModels->Eff CMC Chemistry Manufacturing & Controls Tox->CMC PK->CMC Eff->CMC IND IND Submission CMC->IND Clinical Clinical Trials IND->Clinical

Figure 1: Integrated Workflow for Preclinical Development of NIR-II Imaging Agents

Regulatory Pathways for NIR-II Imaging Agents

Navigating the regulatory landscape requires careful planning from early development stages. For most NIR-II agents, the path to approval follows the Drug Development Toolkit with specific considerations for imaging products.

Regulatory Classification and Strategy

NIR-II fluorescence imaging agents typically fall under the classification of "diagnostic radiopharmaceuticals" or "imaging drugs" despite not being radioactive, due to their functional similarity. The regulatory pathway is primarily through New Drug Application (NDA), with some specific agents potentially qualifying for 505(b)(2) applications if leveraging previously approved molecules (e.g., ICG derivatives) [58]. Early engagement with regulatory agencies through Pre-IND meetings is critical to align on specific requirements for nonclinical studies and clinical trial design.

Essential Regulatory Documentation

Table 2: Key Components of Regulatory Submissions for NIR-II Imaging Agents

Submission Component Key Elements Specific Considerations for NIR-II Agents
Chemistry, Manufacturing, and Controls (CMC) Complete description of composition, manufacturing process, specifications, and stability data Extensive characterization of optical properties (absorption/emission spectra, quantum yield, photostability) [14]
Nonclinical Pharmacology Mechanism of action, proof of concept, dose-response relationships Demonstration of superiority over existing imaging modalities (e.g., NIR-I) through quantitative metrics (SBR, penetration depth) [26] [58]
Nonclinical Toxicology GLP-compliant safety studies in relevant species, toxicokinetics Special attention to organ systems where probe accumulation occurs (typically RES organs); ophthalmic toxicity assessment for intraoperative use [34]
Clinical Development Plan Phase I-II-III protocols, risk-benefit analysis, clinical endpoints For intraoperative agents, pivotal trials typically use endpoints comparing tumor margin identification with and without NIR-II guidance [26]

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful translation of NIR-II imaging technology requires access to specialized materials and instrumentation. The following toolkit encompasses key resources referenced in the search results.

Table 3: Essential Research Reagents and Materials for NIR-II Imaging Development

Category Specific Examples Function/Application Key Characteristics
NIR-II Fluorophores ICG [58], IR-1048 [26], BBTD-cored small molecules [36], VIPI series cyanines [84] Contrast generation for deep-tissue imaging Emission >1000 nm, high quantum yield, appropriate pharmacokinetics
Surface Modification Agents PEGylated phospholipids, poly(maleic anhydride-alt-1-octadecene) (PMAO), bovine serum albumin (BSA) Biocompatibility enhancement, pharmacokinetic modulation Improved solubility, reduced protein corona formation, prolonged circulation
Imaging Systems IR-VIVO [26], LightIR [26], InGaAs camera-based systems Signal detection and quantification Sensitivity in 1000-1700 nm range, real-time imaging capability, compatibility with clinical use
Targeting Moieties Peptides (RGD), antibodies, affibodies Molecular specificity for disease biomarkers High affinity, selectivity, appropriate conjugation chemistry
Model Systems Patient-derived xenografts, orthotopic tumor models, 3D tissue phantoms [26] Efficacy assessment in biologically relevant contexts Recapitulation of human disease pathophysiology, appropriate biomarker expression

G NP Nanoparticle Core Linker Bioconjugation Linker NP->Linker Surface Functionalization Dye NIR-II Fluorophore Dye->Linker Covalent Attachment Stealth Stealth Coating (PEG) Final Final Construct Stealth->Final Target Targeting Ligand Target->Final Linker->Stealth Controlled Orientation Linker->Target Site-Specific Conjugation

Figure 2: Molecular Engineering Strategy for Targeted NIR-II Imaging Probes

The clinical translation of NIR-II fluorescence imaging technology represents a multidisciplinary challenge requiring coordinated advances in materials science, toxicology, regulatory science, and clinical practice. The frameworks and protocols outlined in this document provide a structured approach to addressing the critical biocompatibility, toxicity, and regulatory hurdles. As the field progresses, several emerging trends warrant particular attention: the development of fluorophores with emissions beyond 1500 nm (NIR-IIb) for even better tissue penetration and contrast [36]; the integration of artificial intelligence for enhanced image interpretation and quantification; and the creation of standardized testing protocols specific to NIR-II agents to facilitate regulatory review. With strategic attention to these translational considerations, NIR-II fluorescence imaging is poised to revolutionize clinical practice across multiple medical specialties, particularly in oncology where precise visualization of tumor margins can directly impact patient outcomes. The path to clinical translation, while complex, offers tremendous potential to enhance the precision of diagnostic and therapeutic interventions.

Conclusion

NIR-II fluorescence imaging represents a paradigm shift in optical bioimaging, firmly establishing itself as an indispensable tool for biomedical research with accelerating clinical translation. By leveraging its foundational advantages of deep tissue penetration and high spatial resolution, this technology is revolutionizing applications from intraoperative navigation to the precise evaluation of nanomedicine delivery. While significant progress has been made in fluorophore development and system integration, future efforts must focus on synthesizing brighter, more biocompatible probes, standardizing imaging protocols, and conducting large-scale clinical trials. The convergence of NIR-II imaging with other modalities and its integration into cell therapies and advanced drug delivery platforms promise to further solidify its role as a cornerstone of precision medicine, ultimately improving diagnostic accuracy and therapeutic outcomes for patients.

References