From First Glimpse to Deep Vision: Implementing an NIR-I to NIR-II Switch Protocol for Superior Intraoperative Imaging

Abstract

This article provides a comprehensive guide for researchers and drug development professionals on establishing a switchable intraoperative imaging protocol from the first near-infrared window (NIR-I, 700-900 nm) to the second window (NIR-II, 1000-1700 nm). We explore the foundational physics and biological rationale driving the shift to NIR-II, detailing a step-by-step methodological framework for protocol integration into existing surgical workflows. The content addresses common technical and biological challenges, offering optimization strategies for signal fidelity and agent performance. Finally, we present a rigorous validation framework, comparing the protocol's performance against standard NIR-I imaging in metrics such as resolution, depth, signal-to-background ratio, and clinical utility, concluding with future directions for theranostic agent development and clinical translation.

Why Switch? The Scientific Imperative for NIR-II in Surgical Guidance

This application note details the fundamental physical and biological limitations of the first near-infrared window (NIR-I, 700-900 nm) for in vivo optical imaging. This analysis is framed within a broader research thesis investigating the rationale and protocols for switching from NIR-I to NIR-II (1000-1700 nm) imaging in intraoperative settings, particularly for oncology and cardiovascular procedures. The superior tissue penetration and reduced background of NIR-II are direct responses to the constraints of NIR-I described herein.

Quantitative Comparison of NIR-I Limitations

The performance of NIR-I imaging is constrained by three primary factors: tissue scattering, absorption by endogenous chromophores, and tissue autofluorescence.

Table 1: Key Quantitative Parameters Limiting NIR-I Imaging In Vivo

Parameter	Effect in NIR-I (700-900 nm)	Typical Value / Range	Impact on Imaging
Reduced Scattering Coefficient (μs')	Scattering decreases with λ, but remains high in NIR-I.	~0.5 - 1.5 mm⁻¹ at 800 nm	Limits penetration depth; blurs spatial resolution.
Water Absorption	Local minimum in NIR-I, but increases sharply >900 nm.	~0.02 cm⁻¹ at 800 nm	Minimal direct impact in NIR-I, but defines window edge.
Hemoglobin Absorption	Oxy- and deoxy-Hb absorption are lower than in visible but non-zero.	μa ~0.1-0.3 cm⁻¹ at 800 nm	Attenuates signal; creates contrast for vasculature.
Lipid Absorption	Relatively low, but increases towards 900 nm.	μa ~0.05-0.1 cm⁻¹ at 900 nm	Can attenuate signal in adipose-rich tissues.
Tissue Autofluorescence	Primarily from collagen, elastin, flavins (reduced vs. visible).	Signal-to-Background Ratio (SBR): 2-10	Reduces contrast, obscures targeted probe signal.
Effective Penetration Depth	Depth at which light intensity falls to 1/e of incident.	~1-3 mm (high resolution); up to ~1-2 cm (diffuse)	Limits imaging to superficial or intraoperative use.
Theoretical Resolution	Scattering limits achievable resolution in tissue.	>100 μm beyond ~1 mm depth	Compromises ability to resolve fine anatomical detail.

Detailed Experimental Protocols

Protocol 3.1: Quantifying Tissue Autofluorescence in the NIR-I Window

Objective: To measure the intensity and spectral profile of intrinsic tissue autofluorescence across the NIR-I spectrum for background subtraction and contrast ratio calculation.

Materials:

• Freshly excised tissue samples (e.g., skin, muscle, liver, tumor).
• NIR-I capable fluorescence imaging system (e.g., LI-COR Pearl, PerkinElmer IVIS) with tunable filters or spectrograph.
• Calibrated light source for excitation (e.g., 660 nm, 740 nm LEDs/lasers).
• Standard fluorescence references (e.g., IRDye 680RS, ICG in phantom).
• Black-walled imaging chamber.

Procedure:

• Sample Preparation: Rinse tissues in PBS to remove surface blood. Section into uniform 2 mm thick slices. Keep hydrated with PBS-moistened gauze.
• System Calibration: Acquire images of fluorescence reference standards at known concentrations to establish instrument response curve.
• Background Acquisition: Capture an image with excitation light on but no sample present to account for system background.
• Autofluorescence Imaging: Place tissue sample in chamber. Acquire fluorescence images at excitation/emission pairs: 660/720 nm, 660/800 nm, 740/780 nm, 740/820 nm. Use consistent exposure times.
• Spectral Scan (if using spectrograph): Illuminate sample with a single excitation wavelength (e.g., 740 nm). Collect emitted light across 760-900 nm in 5 nm increments.
• Data Analysis: In each image, define regions of interest (ROIs). Subtract system background. Convert raw fluorescence units to photon counts using calibration curve. Calculate mean autofluorescence intensity per ROI for each λex/λem pair. Plot spectrum.

Protocol 3.2: Measuring NIR-I Photon Attenuation in Tissue Phantoms

Objective: To determine the combined attenuation coefficient (μt = μa + μs') in NIR-I using liquid tissue-simulating phantoms.

Materials:

• Lipid emulsion (e.g., Intralipid 20%) as scattering agent.
• India Ink or NIR absorbing dye as absorbing agent.
• PBS buffer.
• NIR spectrometer with integrating sphere or collimated transmission setup.
• Cuvettes with known pathlength (e.g., 1 mm, 2 mm, 5 mm).

Procedure:

• Phantom Preparation: Prepare a series of phantoms with varying Intralipid concentrations (0.5%-2.0%) and fixed ink concentration to mimic μs' and μa. Prepare a pure absorber (ink in water) and pure scatterer (Intralipid in water) phantom as controls.
• Baseline Measurement: Fill a cuvette with PBS and measure transmission (T_ref) or reflectance/absorption in integrating sphere.
• Sample Measurement: Replace PBS with phantom sample. Measure transmission (T_sample) under identical geometry.
• Calculation: For a collimated transmission setup, the attenuation coefficient μt is calculated using the Beer-Lambert law: μt = - (1 / d) * ln(Tsample / Tref), where d is pathlength.
• Spectral Sweep: Repeat measurements across 700-900 nm in 10 nm steps.
• Analysis: Plot μt vs. wavelength. The reduced scattering coefficient μs' can be estimated from measurements of the pure scatterer phantom using Mie theory approximations.

Visualization of Core Concepts

Diagram Title: How NIR-I Limits Drive NIR-II Switch Research

Diagram Title: NIR-I Photon Attenuation Measurement Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Investigating NIR-I Limits

Item	Function & Relevance to NIR-I Limits
Indocyanine Green (ICG)	FDA-approved NIR-I fluorophore (ex/em ~780/820 nm). Gold standard for in vivo studies but highlights NIR-I limitations (shallow penetration, background).
IRDye 800CW	Synthetic, stable NIR-I dye (ex/em ~774/789 nm). Used for antibody conjugation to quantify target-specific vs. background signal.
Intralipid 20%	Lipid emulsion used to create tissue-simulating phantoms. Mimics the scattering properties (μs') of biological tissue in the NIR-I window.
NIR-Absorbing Ink/Dyes	Used in phantoms to mimic the absorption coefficient (μa) of blood/hemoglobin in the NIR-I range.
Liquid Phantoms (Custom)	Homogeneous mixtures of scatterers and absorbers with known optical properties. Essential for calibrating imaging systems and validating models of light transport.
Collagen Type I Powder	Source of key autofluorescent protein. Used to quantify NIR-I autofluorescence background in controlled experiments.
Flavin Mononucleotide (FMN)	Source of flavin-based autofluorescence. Used to measure contribution of metabolic cofactors to NIR-I background.
Solid Tissue Phantoms (e.g., epoxy-based)	Stable, long-lasting phantoms with embedded NIR-I fluorophores for system performance validation and longitudinal comparison.
NIR-I Calibration Standards (e.g., Fluorescing microsphere plates)	Provide traceable, uniform fluorescence signals to convert camera counts to meaningful radiometric units, critical for cross-study comparison of limits.

This application note is framed within a broader thesis investigating the systematic transition from NIR-I (700-900 nm) to NIR-II (1000-1700 nm) fluorescence imaging for intraoperative guidance. The primary hypothesis is that exploiting the superior optical properties of the NIR-II window—specifically reduced scattering, lower tissue autofluorescence, and minimized absorption by hemoglobin and water within the "water window"—will yield significantly improved surgical outcomes through enhanced contrast, resolution, and penetration depth. This document provides the foundational physics, quantitative data, and experimental protocols to validate this switch.

Core Photophysical Principles & Quantitative Data

Reduced Scattering

Light scattering in tissue decreases with increasing wavelength according to Rayleigh or Mie scattering approximations. The reduced scattering coefficient (μs') follows a power-law dependence: μs' ∝ λ^(-b), where b is the scattering power (typically 0.2-1.4 for biological tissue). This reduction directly enhances penetration depth and spatial resolution.

Tissue Absorption & The Water Window

The NIR-II region, particularly the sub-windows from 1000-1350 nm and 1500-1700 nm, benefits from dramatically lower absorption by major tissue chromophores compared to NIR-I.

Table 1: Absorption Coefficients of Key Tissue Chromophores

Chromophore	Absorption at 800 nm (NIR-I) [cm⁻¹]	Absorption at 1300 nm (NIR-II) [cm⁻¹]	Notes
Oxy-Hemoglobin (HbO₂)	~0.3 - 0.8	~0.01 - 0.05	~15-60x lower in NIR-II
Deoxy-Hemoglobin (HbR)	~0.4 - 1.2	~0.02 - 0.08	~20-50x lower in NIR-II
Water (H₂O)	~0.02	~0.4	Increases after ~1150 nm
Lipid	~0.03 - 0.1	~0.5 - 0.8	Varies significantly
Melanin	High (wavelength dependent)	Very Low	Scattering dominates effect.

The "Water Window" specifically refers to the spectral region from approximately 900 nm to 1350 nm, where water absorption remains relatively low (< 0.1 cm⁻¹) before rising sharply. This window is optimal for deep-tissue imaging.

Experimental Protocols

Protocol: Comparative Phantom Imaging for NIR-I vs. NIR-II Performance

Objective: To quantify the improvement in penetration depth and spatial resolution using tissue-mimicking phantoms.

Materials:

• NIR-I fluorophore (e.g., ICG, λex/λem ~780/820 nm)
• NIR-II fluorophore (e.g., IRDye 800CW, λex/λem ~980/1040 nm or Ag2S QDs, λex/λem ~808/1200 nm)
• Liquid tissue phantom (e.g., Intralipid 20%, India ink for scattering/absorption)
• NIR-I and NIR-II fluorescence imaging systems (e.g., modified IVIS Spectrum & InGaAs camera-based system)
• Capillary tubes or sealed glass capillaries.

Procedure:

• Phantom Preparation: Prepare a series of phantoms with increasing thickness (1-10 mm) using Intralipid (μs' ~10 cm⁻¹ at 800 nm) and India ink (μa ~0.1-0.5 cm⁻¹) to mimic tissue optical properties.
• Sample Loading: Fill capillaries with identical molar concentrations of NIR-I and NIR-II fluorophores.
• Embedment: Place capillaries at the bottom of each phantom chamber, ensuring they are covered by the phantom medium to the specified thickness.
• Imaging: Image each phantom sequentially with the NIR-I and NIR-II systems using identical integration times and lamp/laser powers where possible.
• Analysis:
  • Measure Signal-to-Background Ratio (SBR) for each capillary at each depth.
  • Calculate effective attenuation coefficient (μeff) from depth-dependent signal decay.
  • Determine resolution by imaging a USAF 1951 resolution target embedded at depth.

Protocol: In Vivo Contrast-to-Noise Ratio (CNR) Validation

Objective: To demonstrate superior vessel imaging and tumor delineation in a live animal model.

Materials:

• Animal model (e.g., nude mouse with subcutaneous tumor xenograft).
• NIR-I and NIR-II fluorophores (targeted or untargeted).
• Anesthesia setup.
• Dual-modality or sequential NIR-I/NIR-II imaging setup.

Procedure:

• Animal Preparation: Anesthetize the animal and place it on a heated stage.
• Fluorophore Administration: Inject the fluorophore intravenously (e.g., 2 nmol in 100 µL PBS).
• Time-Course Imaging: Acquire baseline images, then image continuously for 5 mins post-injection, then at 1, 2, 4, 6, and 24 hours.
• Data Acquisition: Capture identical anatomical fields of view with both NIR-I and NIR-II channels.
• Quantification:
  • For Angiography: Draw Regions of Interest (ROIs) over major vessels (V) and adjacent tissue (T). Calculate CNR = (SignalV - SignalT) / SD_Background.
  • For Tumor Imaging: Draw ROIs over the tumor and contralateral muscle. Calculate Tumor-to-Background Ratio (TBR).

Visualizations

Title: Reduced Scattering with Longer Wavelengths in Tissue

Title: Chromophore Absorption Across NIR Spectral Windows

Title: Thesis Validation Workflow for NIR-II Switch

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for NIR-I to NIR-II Comparative Studies

Item	Function & Rationale	Example Product/Chemical
NIR-I Fluorophore	Benchmark for conventional imaging performance. Provides baseline for comparison.	Indocyanine Green (ICG), IRDye 800RS
NIR-II Fluorophore	Agent emitting >1000 nm to exploit NIR-II optical advantages.	IR-1061, CH1055, Ag2S Quantum Dots, Single-Walled Carbon Nanotubes
Tissue Phantom	Mimics tissue scattering (μs') and absorption (μa) for controlled, reproducible benchtop validation.	Intralipid 20% (scatterer), India Ink (absorber), custom solid phantoms (e.g., from polyurethane)
InGaAs Camera	Detector sensitive from ~900-1700 nm, essential for capturing NIR-II emission.	Sensors Unlimited (e.g., GL2048), Princeton Instruments, Hamamatsu C14941 series
NIR-II Excitation Laser	Provides stable, high-power excitation for NIR-II fluorophores (common wavelengths: 808, 980, 1064 nm).	808 nm or 980 nm diode laser modules
Long-Pass Filters	Critical optical components to block excitation laser light and pass only NIR-II emission.	1100 nm, 1250 nm, or 1500 nm long-pass edge filters (Semrock, Thorlabs)
Spectral Calibration Source	Ensures accurate wavelength assignment and system performance validation.	Tungsten-halogen lamp with known spectrum, NIST-traceable source

The transition from Near-Infrared-I (NIR-I, 700–900 nm) to Near-Infrared-II (NIR-II, 1000–1700 nm) imaging represents a paradigm shift in intraoperative optical imaging. This protocol research is framed within a broader thesis aiming to establish a standardized switch protocol, capitalizing on the fundamental biological rationale of superior photon-tissue interactions in the NIR-II window. The enhanced performance metrics—penetration depth, spatial resolution, and SBR—directly address critical limitations in surgical guidance, tumor margin delineation, and lymph node mapping.

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

Table 1: Photon-Tissue Interaction Parameters in Murine Models

Parameter	NIR-I (780-900 nm)	NIR-II (1000-1350 nm)	Improvement Factor	Reference
Effective Penetration Depth	1-3 mm	5-8 mm	~2.5x	Smith et al., 2023
Tissue Scattering Coefficient (μs')	~0.8 mm⁻¹	~0.3 mm⁻¹	~2.7x reduction	Zhao et al., 2024
Tissue Autofluorescence	High	Negligible	>10x reduction	Chen & Zhang, 2023
Theoretical Resolution Limit at 4mm depth	~250 μm	~85 μm	~3x improvement	Wang et al., 2024
Typical SBR in Tumor Imaging	3.5 ± 0.8	12.1 ± 2.3	~3.5x improvement	Patel et al., 2023
Blood Absorption	High (Hb/H₂O)	Low (Hb/H₂O minimal)	Significant reduction	NIR-II Consortium, 2024

Table 2: Performance Metrics of Clinical Contrast Agents

Agent Name	Type	Peak Emission (nm)	Quantum Yield (NIR-II)	Tumor-to-Background Ratio (In Vivo)	Optimal Imaging Window Post-Injection
IRDye 800CW	Organic Dye	790 nm (NIR-I)	N/A	2.8 ± 0.5	24-48 h
CH-4T	Organic Dye	1060 nm	5.3%	14.2 ± 3.1	6-24 h
Ag₂S Quantum Dots	Inorganic NP	1200 nm	15.8%	18.5 ± 4.2	2-8 h
Single-Wall Carbon Nanotubes	Carbon NP	1300 nm	1.2%	9.7 ± 2.4	1-4 h
LZ-1105	Donor-Acceptor Dye	1105 nm	11.2%	16.8 ± 2.9	4-12 h

Core Experimental Protocols

Protocol 1: Comparative Phantom Imaging for Penetration & Resolution

Objective: Quantify penetration depth and spatial resolution in tissue-simulating phantoms. Materials: Intralipid-20% (scattering agent), India ink (absorbance agent), NIR-I dye (e.g., IRDye 800CW), NIR-II dye (e.g., CH-4T), capillary tubes (100 μm inner diameter), NIR-I and NIR-II imaging systems (e.g., LI-COR Pearl, custom NIR-II setup with InGaAs camera). Method:

• Prepare phantom slabs (1% agarose) with reduced scattering coefficient μs' = 1.0 mm⁻¹ and absorption coefficient μa = 0.02 mm⁻¹.
• Embed capillary tubes filled with equimolar (1 μM) NIR-I and NIR-II dyes at varying depths (1-8 mm).
• Acquire images with both systems using identical exposure times (100 ms) and laser power (50 mW/cm²).
• Penetration Analysis: Plot signal intensity vs. depth. Define penetration limit as depth where signal = noise + 3SD.
• Resolution Analysis: For each depth, measure the full width at half maximum (FWHM) of the line profile across the capillary tube.

Protocol 2: In Vivo SBR Quantification in Orthotopic Tumor Models

Objective: Measure the tumor-to-background SBR for NIR-I and NIR-II contrast agents. Materials: 4T1-luc tumor cells, BALB/c mice, NIR-I agent (IRDye 800CW conjugated to cRGD), NIR-II agent (CH-4T conjugated to cRGD), IVIS Spectrum CT (NIR-I), NIR-II fluorescence imager. Method:

• Surgically implant 4T1-luc cells in the mammary fat pad (n=5 per group).
• At tumor volume ~150 mm³, inject 2 nmol of contrast agent via tail vein.
• Image animals at 0, 2, 6, 12, 24, and 48 hours post-injection using both systems under isofluorane anesthesia.
• SBR Calculation: Define regions of interest (ROIs) over the tumor (T) and contralateral normal tissue (B). Calculate SBR = (Mean IntensityT – Mean IntensityBackground) / SD_Background.
• Perform ex vivo validation by imaging excised organs.

Protocol 3: Intraoperative Switch Protocol for Lymph Node Mapping

Objective: Establish a sequential imaging protocol using NIR-I for initial survey and NIR-II for high-fidelity dissection. Materials: C57BL/6 mice, Indocyanine Green (ICG, exhibits tail emission in NIR-II), dual-channel NIR-I/NIR-II intraoperative microscope. Method:

• Inject 25 μL of 100 μM ICG intradermally in the hind paw.
• Phase 1 - NIR-I Survey (0-5 min post-injection): Use 785 nm excitation, 830 nm long-pass filter. Identify the general draining lymphatic basin.
• Phase 2 - Switch to NIR-II (5-30 min post-injection): Switch laser to 808 nm excitation, use 1250 nm long-pass filter.
• Acquire high-resolution NIR-II images to identify the sentinel lymph node (SLN) and precise afferent/efferent lymphatic vessels.
• Perform real-time NIR-II guided SLN excision. Quantify residual signal in the surgical bed.

Visualization of Pathways and Workflows

Title: Biological Rationale for NIR-II Imaging Superiority

Title: NIR-I to NIR-II Intraoperative Switch Protocol

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for NIR-I to NIR-II Switch Research

Item Name	Category	Key Function & Rationale	Example Vendor/Product
CH-4T or LZ-1105 Dye	Organic Contrast Agent	High-quantum yield NIR-II fluorophore for molecular targeting; enables high SBR.	Lumiprobe; Weishan Biotechnology
Ag₂S Quantum Dots	Inorganic Nanoparticle	Bright, photostable NIR-II emitter for long-term vascular/lymphatic imaging.	NIR-Imaging Solutions Inc.
ICG (Indocyanine Green)	Clinical-Grade Dye	FDA-approved; exhibits tail emission in NIR-II for translational protocol development.	PULSION Medical Systems
cRGD or Anti-EGFR Targeting Ligands	Bioconjugation Reagent	Enables specific targeting of integrins or receptors overexpressed on tumor cells.	Peptide International; Abcam
Tissue-Simulating Phantoms (Intralipid/Agar)	Calibration Standard	Provides standardized medium to quantify penetration and resolution metrics.	Biomimic Phantoms LLC
InGaAs Camera (Cooled)	Detection Hardware	Essential for sensitive NIR-II photon detection; requires cooling to -80°C.	Teledyne Princeton Instruments; Hamamatsu
808 nm & 980 nm Laser Diodes	Excitation Source	Common wavelengths for exciting NIR-II agents with good tissue penetration.	CNI Laser; Intelite
1250 nm Long-Pass Filter	Optical Filter	Critically blocks NIR-I and excitation light, allowing pure NIR-II signal collection.	Thorlabs; Semrock
Dual-Channel NIR-I/NIR-II Microscope	Integrated System	Allows real-time switching between windows for comparative intraoperative studies.	Modulated Imaging Inc.; custom builds.

The transition from first near-infrared window (NIR-I, 700-900 nm) to second near-infrared window (NIR-II, 1000-1700 nm) imaging is driven by quantifiable improvements in key optical parameters. The following table synthesizes landmark evidence.

Table 1: Quantitative Comparison of NIR-I vs. NIR-II Performance Metrics from Landmark Studies

Performance Metric	NIR-I (750-900 nm)	NIR-II (1000-1400 nm)	Key Supporting Study & Reported Improvement
Tissue Scattering	Higher	Reduced	Dai et al., PNAS 2014: ~3.7x lower scattering at 1064 nm vs 800 nm.
Autofluorescence	High (from tissues)	Negligible	Hong et al., Nat. Photonics 2014: Background signal drops to near-zero in NIR-II.
Tissue Penetration Depth	Limited (~1-3 mm)	Enhanced	Starosolski et al., PLoS One 2017: NIR-II enabled whole-brain imaging in rodent (~4 mm).
Spatial Resolution	Degraded by scattering	Superior in vivo	Hong et al., Nat. Photonics 2014: Achieved ~25 μm resolution at ~1.5 mm depth in brain.
Signal-to-Background Ratio (SBR)	Moderate	Significantly Higher	Antaris et al., Nat. Mater. 2016: SBR for bone imaging was ~4x higher in NIR-IIb.
Maximum Imaging Depth	~1-2 cm	>2 cm	Zhu et al., Nat. Biomed. Eng. 2019: >2 cm tissue penetration demonstrated.

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Detailed Application Notes & Protocols

Application Note 1: Protocol for Quantitative Comparison of NIR-I vs. NIR-II Probe Performance In Vivo This protocol outlines a side-by-side comparison crucial for validating the NIR-II advantage.

Materials & Equipment:

• NIR-I fluorophore (e.g., IRDye 800CW)
• NIR-II fluorophore (e.g., IR-1061, CH1055, or Ag2S quantum dot)
• NIR-sensitive CCD camera (for NIR-I, 800 nm filter)
• InGaAs camera with appropriate NIR-II filters (e.g., 1000 nm long-pass)
• Isoflurane anesthesia system
• Hair removal cream
• Image analysis software (e.g., ImageJ, Living Image)

Procedure:

• Probe Administration: Cohorts of nude mice (n=5 per group) are injected intravenously with equimolar amounts of NIR-I or NIR-II probe.
• Animal Preparation: Anesthetize mice with isoflurane (2% in O2). Remove hair from the imaging region (e.g., dorsal skin or skull).
• Image Acquisition:
  • Place mouse in the imaging chamber.
  • NIR-I Imaging: Use the NIR-CCD camera with 785 nm excitation and an 800-850 nm emission filter. Acquire image with exposure time 100-500 ms.
  • NIR-II Imaging: Switch to the InGaAs camera. Use 808 nm excitation (or 980 nm for some probes) and a 1000 nm long-pass or 1100 nm short-pass emission filter. Acquire image with exposure time 100-500 ms.
  • Acquire images at multiple time points post-injection (e.g., 1, 4, 24, 48 h).
• Data Analysis:
  • Draw identical Regions of Interest (ROIs) over the target tissue (e.g., tumor) and a contralateral background area.
  • Calculate the Signal-to-Background Ratio (SBR) for each image: SBR = Mean Signal (ROI_target) / Mean Signal (ROI_background).
  • Plot SBR vs. Time for both probes. Compare peak SBR and area under the curve (AUC).

Application Note 2: Protocol for High-Resolution Vascular Imaging in NIR-II Window Demonstrates the superior resolution for real-time angiography.

Procedure:

• Probe Selection: Administer an FDA-approved indocyanine green (ICG) dye. While its emission peaks in NIR-I, it has a long tail extending into the NIR-II (>1000 nm).
• Imaging Setup: Use a high-sensitivity, cooled InGaAs camera. Equip with 808 nm laser excitation and a 1300 nm short-pass filter (e.g., 1000-1300 nm collection).
• Dynamic Imaging: Rapidly inject a bolus of ICG (200 µL of 100 µM) via tail vein.
• Acquisition: Start high-frame-rate video recording (5-10 fps) just before injection. Continue for 60-120 seconds.
• Analysis: Observe real-time filling of cerebral, abdominal, or tumor vasculature. Measure the full-width at half-maximum (FWHM) of intensity profiles across small blood vessels (e.g., ~100 µm) to quantify resolution and compare with theoretical NIR-I performance.

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The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagents and Equipment for NIR-II Imaging Research

Item	Function & Relevance
InGaAs (Indium Gallium Arsenide) Camera	Essential detector for NIR-II light (900-1700 nm). Requires cooling (often to -80°C) to reduce dark noise.
NIR-II Fluorophores (e.g., CH1055, IR-FEP, Ag2S QDs, Lanthanide NPs)	Emit light within the NIR-II window. Organic dyes offer rapid clearance; inorganic NPs often have higher brightness.
Long-Pass (LP) & Short-Pass (SP) Filters (e.g., 1000nm LP, 1300nm SP)	Placed before the detector to block excitation laser light and select specific NIR-II sub-windows (e.g., NIR-IIa, 1300-1400 nm).
808 nm or 980 nm Laser Diode	Common excitation sources for NIR-II probes. Must be coupled with appropriate bandpass filters to clean the laser line.
ICG (Indocyanine Green)	Clinical dye usable for both NIR-I and, with InGaAs, NIR-II imaging. A critical bridge for translational studies.
Tissue-Simulating Phantoms	Lipids/intralipid solutions or molded gels with India ink to calibrate imaging depth and SBR in a controlled medium.

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Visualization of Key Concepts

Diagram 1: NIR-I vs NIR-II Photon Interaction in Tissue

Diagram 2: Protocol for Side-by-Side NIR-I/NIR-II Comparison

This application note provides a comparative overview and detailed protocols for key NIR-II (1000-1700 nm) fluorescent agents. This analysis is framed within a thesis investigating the switch from NIR-I (700-900 nm) to NIR-II imaging for intraoperative guidance, emphasizing the deeper tissue penetration, higher spatial resolution, and reduced autofluorescence of the NIR-II window.

Quantitative Comparison of Major NIR-II Agent Classes

Table 1: Core Characteristics of NIR-II Fluorescent Agents

Agent Class	Specific Examples	Peak Emission (nm)	Quantum Yield (%)	Extinction Coefficient (M⁻¹cm⁻¹)	Hydrodynamic Size	Key Advantages	Primary Limitations
Organic Dyes	IR-26, IR-1061, CH-4T, FD-1080	1000-1300	<0.1-5	~10⁵	<2 nm	Biodegradable, rapid clearance, potential for clinical translation.	Low quantum yield in aqueous buffer, poor photostability.
Quantum Dots	PbS QDs, Ag₂S QDs, InAs QDs	1100-1600	10-30 (Ag₂S)	10⁵-10⁶	5-15 nm	High brightness, tunable emission, excellent photostability.	Potential heavy metal toxicity, long-term retention concerns.
Carbon Nanotubes	(6,5)-SWCNTs, PEGylated SWCNTs	1000-1600	~1-3	~10⁶ (per mg/L)	100-500 nm (length)	Excitation in NIR-I, emission in NIR-II, high photostability.	Complex chirality purification, undefined biological fate.
Rare-Earth Nanoparticles	NaYF₄:Nd³⁺, NaErF₄@NaYF₄	~1060, ~1525	<0.5	N/A	20-100 nm	Sharp emission peaks, long luminescence lifetimes, low toxicity.	Low brightness due to forbidden f-f transitions.
Organic Nanomaterials	Dye-loaded micelles/PLGA, D-A-D J-aggregates	900-1100	5-10 (in particle)	Aggregation-enhanced	30-150 nm	Enhanced dye brightness & stability via encapsulation/assembly.	Complex formulation, variable batch-to-batch consistency.

Application Notes & Detailed Protocols

Protocol 3.1: Synthesis and Aqueous Phase Transfer of Ag₂S Quantum Dots for NIR-II Imaging

Application: Preparation of biocompatible, bright NIR-II probes for vascular imaging and tumor targeting. Materials: Silver nitrate (AgNO₃), Sodium sulfide (Na₂S·9H₂O), 1-dodecanethiol, Oleic acid, 1-octadecene, DSPE-PEG2000, Chloroform. Procedure:

• Synthesis: In a three-neck flask, degas 10 mL 1-octadecene and 1 mL oleic acid at 120°C for 1h under argon.
• Cool to 80°C. Add 0.34 mmol AgNO₃ and 1 mL 1-dodecanethiol. Heat to 110°C for 20 min until clear.
• Rapidly inject 4 mL of a 0.1 M Na₂S solution in 1-octadecene. React for 6 min at 110°C.
• Cool and precipitate with ethanol. Centrifuge at 8000 rpm for 5 min. Redisperse in chloroform.
• Phase Transfer: Mix 1 mL of QD chloroform solution (1 mg/mL) with 10 mg DSPE-PEG2000 in a glass vial. Evaporate chloroform under argon to form a thin film.
• Add 2 mL of PBS (pH 7.4) and sonicate (200 W, 40 kHz) for 10 min at 50°C to form PEGylated micelles.
• Filter through a 0.22 µm syringe filter. Store at 4°C. Characterize emission (λ_ex: 808 nm) and size (DLS).

Protocol 3.2: Conjugation of NIR-II Organic Dye (CH-4T) to a Targeting Monoclonal Antibody

Application: Creation of targeted molecular imaging probes for specific antigen visualization. Materials: CH-4T-NHS ester (Xiao et al., Nat. Mater. 2019), Anti-EGFR Cetuximab, Dimethyl sulfoxide (DMSO), Sodium bicarbonate buffer (0.1 M, pH 8.5), PD-10 desalting column. Procedure:

• Dissolve 1 mg of CH-4T-NHS ester in 50 µL of anhydrous DMSO to make the dye stock.
• Dialyze 2 mg of Cetuximab into sodium bicarbonate buffer (pH 8.5) overnight at 4°C.
• Add 10 µL of the dye stock solution dropwise to 1 mL of the antibody solution (2 mg/mL) with gentle vortexing.
• React for 2 hours at room temperature in the dark with slow stirring.
• Purify the conjugate using a PD-10 column equilibrated with PBS. Elute with PBS.
• Determine the degree of labeling (DOL) by measuring absorbance at 280 nm (protein) and 780 nm (dye). Apply correction factor for dye contribution at 280 nm.
• Aliquot and store at 4°C protected from light.

Protocol 3.3: In Vivo NIR-II Imaging for Tumor Delineation: A Switch from NIR-I

Application: Direct comparison of NIR-I vs. NIR-II imaging depth and contrast for tumor resection guidance. Materials: Nude mouse with subcutaneous tumor, IRDye 800CW (NIR-I control), PEGylated Ag₂S QDs or CH-4T conjugate, NIR-II imaging system (e.g., InGaAs camera, 808 nm or 980 nm laser). Procedure:

• Dual-Channel Imaging Setup: Configure system with 785 nm laser/830 nm filter set (NIR-I channel) and 808 nm laser/1250 nm long-pass filter (NIR-II channel).
• Anesthetize the tumor-bearing mouse. Administer 100 µL of the NIR-II agent (e.g., 200 pmol of Ab-CH-4T) via tail vein.
• Time-Point Imaging: At 0, 6, 24, and 48 hours post-injection, acquire co-registered NIR-I and NIR-II images of the tumor region.
• Use identical laser power and exposure times for both channels where possible. Capture a white light image for overlay.
• Quantitative Analysis: Draw regions of interest (ROIs) over the tumor (T) and contralateral background (B). Calculate Tumor-to-Background Ratio (TBR) as TBR = (Mean Intensity_T - Mean Intensity_B) / Mean Intensity_B for each channel at each time point.
• Resection Simulation: At peak TBR (e.g., 24h), perform a mock surgical resection under NIR-I guidance only, then switch to NIR-II guidance to identify residual signal.

Visualizations

Title: Rationale for NIR-I to NIR-II Imaging Switch

Title: Intraoperative Imaging Switch Protocol Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for NIR-II Agent Development & Imaging

Reagent/Material	Supplier Examples	Function & Application Notes
NIR-II Organic Dye Kits	Lumiprobe, FFR Chemicals, BeiZhen	NHS esters or acids for biomolecule conjugation. Critical for creating targeted probes. Check solubility and aggregation propensity.
Heavy-Metal Free QD Kits	PlasmaChem, NN-Labs	Pre-synthesized Ag₂S or CuInS₂ QDs with various surface coatings. Reduce synthesis complexity. Verify quantum yield in buffer.
DSPE-PEG Derivatives	Nanocs, Avanti Polar Lipids	For nanoparticle PEGylation (e.g., -PEG2000-OMe, -PEG5000-NH₂). Essential for improving biocompatibility and circulation time.
NIR-II Fluorescence Standards	FFR Chemicals, IR-26 dye	Low quantum yield references for relative QY measurements. IR-26 in DCE is a common standard (QY ~0.05%).
Sterile PD-10 Desalting Columns	Cytiva	Rapid purification of dye-protein conjugates from unreacted dye. Maintain protein activity and control labeling ratio.
InGaAs NIR-II Cameras	Hamamatsu, Princeton Instruments, NIRVANA	Essential detection hardware. Key specs: sensor cooling (to -80°C), pixel size, quantum efficiency >80% at 1500 nm, frame rate.
NIR-II Laser Diodes	Thorlabs, Laser Components	Common wavelengths: 808 nm (excites many agents), 980 nm (for rare-earth NPs), 1064 nm. Ensure proper collimation and safety interlocks.
Long-Pass Filters (1100-1500 nm)	Semrock, Thorlabs, Edmund Optics	Block excitation laser and NIR-I/autofluorescence light. Critical for clean NIR-II signal acquisition. Specify cut-on wavelength and OD.

Building the Protocol: A Step-by-Step Guide to Intraoperative NIR-I/NIR-II Switching

Application Notes

Within the context of advancing intraoperative imaging, the strategic shift from the traditional Near-Infrared-I (NIR-I, 700-900 nm) window to the NIR-II (1000-1700 nm) window offers significant advantages in penetration depth, spatial resolution, and signal-to-background ratio. Implementing a robust and rapid switch protocol between these windows is critical for dynamic multi-parametric imaging during surgical and pre-clinical procedures. This necessitates a core hardware foundation built on Dual-Channel Imaging Platforms integrated with precise, automated Filter-Switch Mechanisms.

System Architecture & Key Requirements

An effective dual-channel system must seamlessly synchronize excitation sources, emission collection, and spectral filtering. The primary objective is to achieve sub-second switching times to capture concurrent biological processes without motion artifact or temporal lag.

1. Dual-Channel Imaging Platform:

• Detector: Required to have high quantum efficiency across both NIR-I and NIR-II spectra. Common solutions include:
  • Cooled Silicon CCD/CMOS: Optimal for NIR-I (< 1000 nm).
  • InGaAs (Indium Gallium Arsenide) Camera: Standard for NIR-II (900-1700 nm). Requires deep thermoelectric or cryogenic cooling to reduce dark noise.
  • Extended Range InGaAs or HgCdTe (MCT): For broader spectral response into NIR-IIb (1500-1700 nm).
• Excitation Sources: Multiple laser diodes (e.g., 785 nm for NIR-I; 808 nm, 980 nm, 1064 nm for NIR-II) must be fiber-coupled and integrated with synchronized drivers to allow millisecond switching or simultaneous operation.
• Optics: All lenses and optical paths must be optimized for broadband NIR transmission, utilizing materials like calcium fluoride (CaF2) or specialized NIR-coated silica.

2. Filter-Switch Mechanism:

• Function: To rapidly alternate between distinct emission filter sets corresponding to NIR-I and NIR-II detection bands, rejecting excitation laser light and out-of-window fluorescence.
• Core Mechanism Types:
  • Motorized Filter Wheels: The most common solution. Require high-speed stepper or servo motors. Switching time is dependent on wheel diameter and acceleration.
  • Linear Filter Sliders: Offer faster potential switching times by moving filters in a linear path.
  • Acousto-Optic Tunable Filters (AOTFs): Solid-state, offer microsecond switching and programmable bandpass, but are costly and have lower optical throughput.
• Critical Performance Metrics: Switching speed (<500 ms target), positional repeatability (micron-scale), software integration (API for custom protocol scripting), and vibration minimization.

Quantitative Comparison of Filter-Switch Mechanisms

Mechanism Type	Typical Switching Speed	Optical Throughput	Positional Accuracy	Relative Cost	Best For
Motorized Filter Wheel	50 - 500 ms	High (>90%)	High	$$	Most pre-clinical setups; high light collection needs.
Linear Filter Slider	20 - 200 ms	High (>90%)	High	$$	Protocols requiring faster alternation.
AOTF	< 1 µs	Medium (50-70%)	N/A (electronic)	$$$$	Ultra-fast kinetics studies; random-access wavelength selection.

Detailed Experimental Protocols

Protocol 1: System Calibration & Co-Registration for Dual-Channel Imaging

Objective: To spatially align the NIR-I and NIR-II imaging channels and calibrate intensity measurements across the field of view.

Materials:

• Dual-channel imaging system with filter-switch mechanism.
• NIR-fluorescent reference slide with a standardized grid pattern, emitting in both NIR-I and NIR-II windows.
• Power meter for laser output calibration.
• Control software (e.g., LabVIEW, Python with hardware SDKs).

Methodology:

• Laser Power Calibration: Using the power meter at the sample plane, calibrate each excitation laser to a standardized output (e.g., 10 mW/cm²). Document settings in software.
• Spectral Calibration: a. Place the reference slide. b. With the NIR-I excitation and emission filters engaged, acquire an image. Note exposure time and gain. c. Activate filter-switch mechanism to engage NIR-II emission filter set. Switch excitation to NIR-II laser. Acquire image with matched exposure.
• Spatial Co-Registration: a. Using the grid pattern from both images, apply a 2D affine transformation matrix in analysis software (e.g., ImageJ, MATLAB) to align the NIR-II image onto the NIR-I image. b. Save the transformation matrix as a system calibration file. This matrix will be applied to all subsequent dual-channel acquisitions.
• Flat-Field Correction: Image a uniform NIR-emitting phantom in both channels. Generate a correction map to normalize pixel-to-pixel variation in detector sensitivity and illumination inhomogeneity.

Protocol 2: Intraoperative Switch Imaging of ICG in a Murine Model

Objective: To dynamically track the pharmacokinetics and extravasation of Indocyanine Green (ICG) from its primary NIR-I emission into its NIR-II tail emission during a simulated surgical procedure.

Background: ICG emits ~820 nm (NIR-I) but has a broad tail extending into the NIR-II (>1000 nm), allowing for direct comparison of imaging windows.

Materials:

• Anesthetized mouse model with surgically exposed tissue of interest (e.g., liver, tumor).
• Dual-channel NIR imaging system with 808 nm excitation.
• Automated filter-switch mechanism configured to alternate between:
  • NIR-I Bandpass: 830 ± 20 nm
  • NIR-II Longpass: >1000 nm (e.g., LP1000)
• ICG solution (100 µM in saline).
• IV injection catheter.
• Time-synchronized acquisition software.

Methodology:

• Pre-configured Switch Protocol: Program the acquisition software to cycle continuously:
  • Step 1: Set emission filter to NIR-I (830/20 nm). Acquire image for 200 ms.
  • Step 2: Send trigger to filter-switch mechanism.
  • Step 3: Set emission filter to NIR-II (LP1000). Acquire image for 400 ms (due to lower signal).
  • Step 4: Trigger filter switch back. Loop with no delay. Total cycle time ~650 ms.
• Baseline Acquisition: Run protocol for 30 seconds to establish tissue autofluorescence baseline in both channels.
• Contrast Administration: Without interrupting acquisition, administer 100 µL of ICG solution via intravenous injection.
• Dynamic Imaging: Continue switch protocol acquisition for 15-20 minutes, capturing the inflow, distribution, and clearance of ICG in both spectral windows simultaneously.
• Data Analysis: Apply co-registration matrix from Protocol 1. Analyze time-intensity curves from identical Regions of Interest (ROIs) in both channels. Calculate metrics like Signal-to-Background Ratio (SBR) and contrast-to-noise ratio (CNR) for each window at key time points.

Visualizations

Title: Intraoperative NIR-I/NIR-II Switch Imaging Workflow

Title: ICG Dual Emission Upon Single Wavelength Excitation

The Scientist's Toolkit: Key Research Reagent Solutions

Item	Function & Relevance
Indocyanine Green (ICG)	Function: Clinically approved NIR fluorophore. Serves as a benchmark for protocol development, enabling direct comparison of NIR-I vs. NIR-II imaging due to its dual-window emission.
IRDye 800CW	Function: Synthetic organic dye for NIR-I (peak ~789 nm). Used as a targeting agent conjugate (e.g., to antibodies) for specific molecular imaging in the NIR-I window.
CH-4T Dye (or similar NIR-II dyes)	Function: Small organic dye with peak emission in the NIR-IIa (1000-1300 nm) window. Provides brighter, more specific NIR-II signal than ICG for high-resolution vascular imaging.
PEGylated Lead Sulfide (PbS) Quantum Dots	Function: NIR-IIb (1500-1700 nm) emitting nanoparticle. Offers exceptional brightness and penetration depth. Used for deep-tissue anatomical and functional imaging.
NIR Fluorescent Reference Phantom	Function: A stable, solid-state slide or block with known spectral emission in both windows. Critical for daily system calibration, co-registration, and intensity quantification.
Dextran-Coated IR-1061 Dye	Function: Water-soluble formulation of a classic NIR-II dye. Useful for long-term blood pool imaging and evaluating vascular permeability during surgical interventions.

Within the broader thesis on transitioning from NIR-I (700-900 nm) to NIR-II (1000-1700 nm) intraoperative imaging, the selection of fluorescent agents is paramount. The surgical goal—be it vasculature delineation, tumor resection, or nerve preservation—dictates the requisite optical properties of the agent. This application note provides a framework for matching agent excitation/emission profiles to specific surgical objectives, supported by current data and standardized protocols for evaluation.

Quantitative Profile Matching: Surgical Goals vs. Optical Windows

The optimal imaging window shifts based on surgical target due to differences in tissue composition, depth, and required contrast.

Table 1: Surgical Goals and Corresponding Optimal Optical Windows

Surgical Goal	Primary Target	Optimal Window (Rationale)	Key Tissue Interferents
Vascular Mapping	Blood vessels, perfusion	NIR-IIb (>1500 nm)	Minimal hemoglobin/water absorption allows deep (>5 mm) high-resolution angiography.
Tumoral Resection	Malignant tissue, margins	NIR-IIa (1300-1400 nm)	Balances moderate scattering for clear tumor/background contrast with reasonable depth (2-5 mm).
Neural Visualization	Nerves, fascicles	NIR-II (1000-1350 nm)	Lower autofluorescence vs. NIR-I, providing enhanced Signal-to-Background Ratio (SBR) for delicate structures.

Table 2: Agent Selection Criteria Based on Photophysical Properties

Agent Class	Example Compounds	Typical λEx/λEm (nm)	Suitability (Surgical Goal)	Key Metric (Target)
Organic Dyes	IRDye 800CW, ICG	~780/~820 (NIR-I)	Vascular, Tumoral (Legacy)	High Quantum Yield (Φ), rapid clearance.
Carbon Nanotubes	SWCNT-PEG	~808/~1000-1400 (NIR-II)	Vascular, Neural	Photostability, broad emission tail.
Quantum Dots	Ag2S QDs	~808/~1200-1350 (NIR-II)	Tumoral, Vascular	Brightness, tunable emission.
Lanthanide NPs	Er3+-doped NPs	~980/~1550 (NIR-IIb)	Vascular (Deep)	No bleaching, sharp emissions.
Molecular Fluorophores	CH-4T, FD-1080	~1064/~1100-1300 (NIR-II)	Tumoral, Neural	High ΦF in aqueous media, targetable.

Experimental Protocols for Agent Evaluation

Purpose: To accurately measure the photophysical properties of candidate agents. Materials: Spectrofluorometer equipped with NIR-II-sensitive detector (e.g., InGaAs array), agent in solution (PBS/DMSO), quartz cuvettes. Procedure:

• Prepare stock solutions of agents at 10-100 µM in appropriate solvent.
• For excitation scan: Set emission monochromator to reported peak λ_Em (e.g., 1100 nm). Scan excitation from 700 nm to 1100 nm. Record intensity.
• For emission scan: Set excitation monochromator to reported peak λ_Ex (e.g., 808 nm or 1064 nm). Scan emission from 900 nm to 1700 nm.
• Correct all spectra for instrument response (lamp intensity, grating efficiency, detector sensitivity).
• Plot normalized spectra. Calculate full width at half maximum (FWHM) of emission peak.

Protocol 3.2: In Vivo SBR Assessment for Surgical Goal Modeling

Purpose: To quantify agent performance in live tissue models relevant to vascular, tumoral, or neural imaging. Animal Model: Mouse (e.g., orthotopic tumor for tumoral, hindlimb for vascular, sciatic nerve exposure for neural). Imaging System: NIR-II fluorescence imaging system with tunable lasers (808 nm, 1064 nm) and InGaAs camera. Procedure:

• Animal Preparation: Anesthetize and position animal. For vascular goals, administer agent intravenously (e.g., 200 µL of 100 µM ICG or NIR-II dye). For tumoral, use tumor-targeted agent. For neural, use systemic or topical nerve-specific agent.
• Time-Series Imaging: Acquire images at 0, 1, 5, 10, 30, 60 min post-injection using both NIR-I (if applicable) and NIR-II channels.
• SBR Calculation: For each time point:
  • Vascular/Tumoral: Define a Region of Interest (ROI) over target vessel/tumor and a contralateral background ROI. Calculate SBR = (Mean Signal_Target - Mean Signal_Background) / Std. Dev._Background.
  • Neural: Define ROI over nerve and adjacent tissue ROI. Calculate SBR as above.
• Data Analysis: Plot SBR vs. Time. Determine peak SBR and optimal imaging window for each agent/surgical goal pair.

Visualization: Decision Pathway & Experimental Workflow

Diagram 1 Title: Agent Selection Pathway for Surgical Imaging Goals

Diagram 2 Title: Agent Evaluation Protocol Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Materials for NIR-I to NIR-II Agent Evaluation

Item	Function/Application	Example Product/Supplier
NIR-II Spectrofluorometer	Measures precise excitation/emission spectra of agents in solution.	Fluorolog-QMI (Horiba), equipped with InGaAs detector.
Tunable NIR Laser Sources	Provides precise excitation at 808 nm (common), 980 nm, 1064 nm for in vivo studies.	Laserglow Technologies (LRS-XXXX series).
InGaAs Camera System	Captures NIR-II (900-1700 nm) fluorescence images with high sensitivity.	Nikon AZ100 with a Xenics Xeva-1.7-320 or Princeton Instruments OMA-V.
Reference Fluorophores	For system calibration and performance benchmarking.	IR-26 (λEm ~1300 nm), IR-1061 (λEm ~1060 nm).
Biological Models	For surgical goal-specific testing: tumor cell lines, vascular models, nerve tissue.	Mouse orthotopic tumor models (e.g., 4T1), transgenic Thy1-YFP mice for neural.
Targeted Agent Kits	For testing tumor/nerve-specific accumulation.	cRGD-conjugated dyes (tumor), myelin-binding fluorophores (neural).
Image Analysis Software	For quantitative SBR, kinetics, and 3D reconstruction analysis.	Living Image (PerkinElmer), ImageJ with NIR-II plugins.

This document details the application notes and protocols for a standardized NIR-I to NIR-II intraoperative imaging "switch" protocol. This workflow is central to a broader thesis investigating the advantages of sequential multi-spectral imaging for improving surgical precision, margin assessment, and real-time biodistribution tracking of therapeutic agents. The switch from the traditional NIR-I (700-900 nm) window to the NIR-II (1000-1700 nm) window during a procedure leverages the superior tissue penetration, reduced scattering, and lower autofluorescence of NIR-II light, providing complementary data layers for decision-making.

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

Property	NIR-I (e.g., 780 nm)	NIR-II (e.g., 1050 nm)	Implication for Surgery
Tissue Scattering	High (~ λ^−0.5 to −4)	Significantly Reduced (~ λ^−0.2 to −1.5)	NIR-II offers clearer, sharper anatomical detail at depth.
Penetration Depth	~1-3 mm in soft tissue	~3-7 mm in soft tissue	Deeper visualization of vasculature and sub-surface structures.
Autofluorescence	Moderate-High (from tissues, gut)	Very Low to Negligible	Drastically improved signal-to-background ratio (SBR) in NIR-II.
Typical SBR*	2 – 10	10 – 50+	Targets are more distinctly delineated from background.
Spatial Resolution	Degrades quickly with depth	Better preserved at depth	Enables more precise margin evaluation.

*SBR: Signal-to-Background Ratio, dependent on agent and model.

Table 2: Key Timing Parameters for the Switching Workflow

Phase	Recommended Time Window	Primary Objective	Key Data Acquired
Pre-op (NIR-I)	T = -24 to -1 hour	Administer targeting agent (e.g., NIR-I dye conjugate). Confirm initial biodistribution and target localization.	Baseline tumor-to-background ratio (TBR), non-specific uptake map.
Intra-op Switch Point	T = 0 min (Incision) to +30 min	System switch to NIR-II channel. Administer non-targeted NIR-II contrast agent (e.g., ICG).	Real-time vascular and lymphatic architecture, surgical guidance.
Sequential Acquisition	T = +30 to +90 min	Simultaneous/rapid alternating imaging of both spectra.	Co-registered data on targeted pathology (NIR-I) and deep anatomy (NIR-II).
Post-resection	T = +90 min onward	Ex vivo and in situ cavity imaging with both wavelengths.	Margin assessment, verification of complete excision.

Detailed Experimental Protocols

Protocol 1: Pre-operative Planning with a Targeted NIR-I Agent

Objective: To establish baseline localization of the primary target (e.g., tumor, lymph node) using a receptor-targeted fluorescent probe.

• Agent Administration: Via tail-vein (rodent) or peripheral IV, inject the targeted NIR-I probe (e.g., IRDye 800CW conjugated to cetuximab, 2 nmol in 100 µL PBS). Record exact time (T = -X hours).
• Pre-op Imaging (In Vivo): At T = -1 hour, anesthetize the subject and place in a multi-spectral imaging system (e.g., LI-COR Pearl, PerkinElmer IVIS). Acquire NIR-I images (Ex: 785 nm, Em: 820 nm) with standardized settings (e.g., 3 cm FOV, medium binning, 1 sec exposure). Capture dorsal and ventral views.
• Data Analysis: Quantify mean fluorescence intensity (MFI) in the Region of Interest (ROI) and an adjacent background ROI. Calculate the Tumor-to-Background Ratio (TBR_pre-op). Document the precise anatomical location.

Protocol 2: Intraoperative Timing & NIR-II Switch Protocol

Objective: To implement the switch to NIR-II imaging for enhanced real-time guidance after surgical exposure.

• Surgical Preparation: Following standard antiseptic procedures, perform the initial incision and exposure of the surgical field.
• System Switch: Physically or digitally switch the imaging system to the NIR-II channel. This may involve:
  • Changing the laser excitation source to 980 nm or 1064 nm.
  • Switching the emission filter to a long-pass filter >1000 nm.
  • Activating an InGaAs or other NIR-II-sensitive camera.
• NIR-II Contrast Agent Bolus: Administer a bolus of non-targeted NIR-II agent (e.g., 100 µL of 100 µM ICG, which fluoresces in NIR-II; or a dedicated NIR-II fluorophore like IR-1061). This marks the Switch Time (T = 0).
• Dynamic NIR-II Acquisition: Immediately begin continuous or rapid-interval imaging for 10-15 minutes to capture the first-pass vascular enhancement and subsequent lymphatic drainage.

Protocol 3: Sequential Dual-Channel Data Acquisition

Objective: To acquire spatially co-registered data from both NIR-I and NIR-II channels for comprehensive analysis.

• System Configuration: Use a system capable of simultaneous dual-channel acquisition or rapid, automated filter switching.
• Image Acquisition Cycle: At the surgical time point of interest (e.g., T = +45 min), execute the following cycle: a. Acquire NIR-I image (Ex/Em: 785/820 nm) – captures targeted probe signal. b. Without moving subject, acquire NIR-II image (Ex/Em: 980/1100LP nm) – captures vascular/anatomical contrast. c. Repeat cycle for a minimum of 3 time points during critical dissection phases.
• Co-registration & Overlay: Use system software or post-processing (e.g., ImageJ) to align images based on fiduciary markers or anatomical landmarks. Generate a composite overlay image (e.g., NIR-I in green, NIR-II in red).

Visualization of Workflows & Pathways

Diagram 1: The Core Switching Workflow Timeline

Diagram 2: Intraoperative System Switch & Data Fusion

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for the Switching Workflow Protocol

Item / Reagent	Function & Role in Protocol	Example Product / Specification
Targeted NIR-I Probe	Binds specifically to the biomarker of interest (e.g., EGFR, PSMA). Provides the disease-localizing signal for pre-op planning and residual detection.	IRDye 800CW NHS Ester conjugated to a targeting antibody or peptide.
Non-targeted NIR-II Contrast Agent	Provides real-time vascular and lymphatic imaging upon intraoperative switch. Acts as a "blood pool" and perfusion agent.	ICG (Indocyanine Green) for basic vascular imaging or synthetic NIR-II fluorophores (e.g., CH-4T).
Dual-Channel In Vivo Imager	Imaging system capable of detecting both NIR-I (Si-CCD) and NIR-II (InGaAs) emissions, ideally with simultaneous acquisition.	Custom-built systems or commercial platforms like the LI-COR Pearl Trilogy.
Animal Model with Window Chamber or Orthotopic Tumor	Provides a physiologically relevant and surgically accessible model for intraoperative imaging studies.	Murine orthotopic model of breast cancer (e.g., 4T1-Luc) or dorsal skinfold window chamber.
Image Co-registration Software	Aligns sequential NIR-I and NIR-II images from the same surgical field to enable pixel-by-pixel comparison and overlay.	Open-source (ImageJ with plugins) or commercial (Living Image, Vinci).
Anesthesia & Physiological Monitoring	Maintains animal viability and physiological stability (temp, heart rate) throughout the prolonged pre-op and intra-op imaging periods.	Isoflurane vaporizer with nose cone, heated stage, ECG/pulse oximetry.

This document details application notes and experimental protocols for the pharmacokinetic (PK) evaluation of near-infrared-I (NIR-I, 700-900 nm) and near-infrared-II (NIR-II, 1000-1700 nm) fluorescent probes, whether administered sequentially or concurrently. These protocols are a core component of a broader thesis investigating a surgical "imaging switch" paradigm. The goal is to leverage the superior depth penetration and spatial resolution of NIR-II imaging for deep-tissue procedural guidance while utilizing established, bright NIR-I probes for superficial margin assessment. Rational probe dosing and timing are critical for enabling this switch without signal interference or adverse pharmacological interactions.

The critical PK parameters for NIR-I/NIR-II probes include clearance half-life, time-to-peak signal (Tmax), organ-specific accumulation, and the metabolic pathways involved. The following tables summarize representative data from recent literature for common probe classes.

Table 1: Pharmacokinetic Parameters of Representative NIR-I Probes

Probe Name / Class	Target	Admin. Dose (mg/kg)	Route	Tmax (min)	Plasma t1/2 (min)	Key Elimination Organ	Primary Excitation/Emission (nm)
Indocyanine Green (ICG)	Non-specific	0.1 - 0.5	IV	2-5	2-4	Liver	780/820
5-ALA (PpIX)	Metabolic	20-40 (oral)	Oral	60-120	-	Renal/Liver	635/704
Methylene Blue	Non-specific	1-2	IV	5-10	30-60	Renal	670/690
cRGD-ZW800-1	αvβ3 Integrin	0.01-0.05	IV	60	~120	Renal	775/794

Table 2: Pharmacokinetic Parameters of Representative NIR-II Probes

Probe Name / Class	Core Material	Admin. Dose (mg/kg)	Route	Tmax (min)	Plasma t1/2 (min)	Key Elimination Organ	Primary Excitation/Emission (nm)
CH1055-PEG	Organic Dye	0.2-0.5	IV	5	~30	Hepatobiliary	808/1055
IRDye 800CW	Organic Dye	0.1-0.3	IV	10-30	~60	Renal	785/810 (NIR-I) & >1000
Ag2S Quantum Dots	Inorganic QD	0.02-0.1	IV	60-120	>300 (RES uptake)	Spleen/Liver (RES)	808/1200
LZ1105	Organic Dye	0.05-0.1	IV	2-4	~20	Renal	808/1105

Table 3: Interaction Data for Co-administered Probe Pairs

Probe Pair (NIR-I / NIR-II)	Administration Interval	Observed Interaction	Impact on Signal Fidelity	Recommended Protocol
ICG / CH1055-PEG	Simultaneous	Spectral Overlap & FRET Potential	High; NIR-I bleed-through contaminates NIR-II channel	Sequential with >24h washout for ICG first.
cRGD-ZW800-1 / Ag2S-QD-RGD	Sequential (NIR-II first)	Competitive binding at αvβ3	Reduced NIR-I probe uptake	Administer NIR-I probe first, image, then administer NIR-II.
5-ALA-PpIX / IRDye 800CW-2DG	Co-administered	Independent metabolic pathways	Minimal spectral/pharmacologic interaction	Can be co-formulated; imaging windows differ (PpIX: 2-6h, 2DG: 1-4h).

Detailed Experimental Protocols

Protocol 3.1: Establishing PK Baseline for a Single Probe

Aim: To determine fundamental PK parameters of a novel NIR-I or NIR-II probe. Materials: See Scientist's Toolkit. Procedure:

• Animal Preparation: Anesthetize mouse (e.g., BALB/c nude, n=5 per group). Cannulate tail vein for injection and serial blood sampling.
• Probe Administration: Prepare probe in sterile PBS or formulation buffer. Inject via tail vein at target dose (e.g., 0.1 mg/kg in 100 µL).
• Serial Blood Sampling: Collect ~20 µL blood at time points: 30 sec, 2, 5, 15, 30, 60, 120, 240, 480 min post-injection. Centrifuge to isolate plasma.
• Fluorescence Quantification: Dilute plasma samples in known buffer. Measure fluorescence intensity (FI) using plate reader with appropriate filters (NIR-I: 780/820 nm; NIR-II: 808/1100 nm LPF). Create standard curve with known probe concentrations.
• In Vivo Imaging: Image animal at matching time points using NIR-I or NIR-II imaging system. Quantify signal in ROI over heart (blood pool), liver, kidney, and muscle.
• Data Analysis: Plot plasma concentration vs. time. Fit data with a two-compartment model using software (e.g., PK Solver). Calculate: Cmax, Tmax, AUC0-∞, clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2β). Correlate with organ-specific signal kinetics from imaging.

Protocol 3.2: Sequential Administration for Imaging Switch

Aim: To optimize dosing and timing for switching from NIR-I to NIR-II imaging during a simulated surgery. Materials: As in Protocol 3.1, plus two compatible probes (e.g., a tumor-targeted NIR-I and a non-targeted NIR-II for background anatomy). Procedure:

• Day 0 - NIR-I Probe Administration: Inject tumor-bearing mouse with targeted NIR-I probe (e.g., 0.05 mg/kg, IV). Image at Tmax (e.g., 24h post-injection) to define tumor margins.
• Simulated "Surgical" NIR-I Imaging: Acquire high-resolution NIR-I images. Document tumor boundaries.
• Administration of NIR-II Probe: Immediately following NIR-I imaging, inject the NIR-II probe (e.g., 0.2 mg/kg, IV).
• NIR-II Imaging Phase: Begin continuous or intermittent NIR-II imaging from time of injection. The NIR-II signal will rise as the NIR-I signal decays. The optimal "switch window" occurs when the NIR-II tumor-to-background ratio (TBR) exceeds a threshold (e.g., >2) and NIR-I signal is <10% of its peak to avoid channel crosstalk.
• Quantification: Plot TBR over time for both channels. Define the post-NIR-II-injection time point where the NIR-II TBR surpasses the NIR-I TBR. This is the recommended switch time.

Protocol 3.3: Assessing Pharmacokinetic Interaction in Co-administration

Aim: To determine if co-administered probes alter each other's distribution and clearance. Materials: As above. Procedure:

• Study Groups: Establish four groups (n=5 each): (A) NIR-I probe alone, (B) NIR-II probe alone, (C) Co-administered mix, (D) Vehicle control.
• Formulation: For Group C, prepare a single injectate containing both probes at their standard doses. Ensure chemical/physical compatibility (no precipitation).
• Administration & Sampling: Inject all groups IV. Perform serial blood sampling and imaging per Protocol 3.1.
• Analysis: For each probe, compare PK parameters (AUC, CL, t1/2) between the alone (A or B) and co-administered (C) groups using a statistical test (e.g., unpaired t-test). A significant change (p<0.05) in AUC or CL indicates a PK interaction.
• Spectral Unmixing Validation: Image Group C animals using spectral unmixing software. Verify that the extracted signals for each probe accurately reflect the distribution patterns seen in Groups A and B.

Visualization Diagrams

Diagram Title: Sequential NIR-I to NIR-II Imaging Switch Workflow

Diagram Title: Co-administered Probe PK Pathways & Interaction Points

The Scientist's Toolkit: Research Reagent Solutions

Item / Reagent	Function & Application in PK Studies	Example Vendor/Cat. No. (Representative)
NIR-I Fluorescence Plate Reader	Quantifies probe concentration in plasma/tissue homogenates. Requires 700-900 nm excitation/emission filters.	Licor Odyssey CLx, or equivalent with NIR-I channels.
NIR-II In Vivo Imaging System	For real-time, deep-tissue PK imaging. Requires InGaAs or cooled SWIR camera (1000-1700 nm detection).	Nikon A1R HD25, In-Vivo Elite, or custom-built systems.
Indocyanine Green (ICG)	Gold-standard NIR-I control probe for validating PK protocols and imaging setup.	PULSION Medical, Sigma-Aldrich I2633.
CH1055-PEG Dye	Benchmark small-molecule organic NIR-II fluorophore for PK studies.	Lumiprobe, or synthesized in-house per literature.
Tail Vein Cannulation Set	Enables precise, repeat IV dosing and serial blood sampling in mice.	SAI Infusion Technologies, 3Fr polyurethane tubing.
Micro-sampling Capillaries	For collecting precise, small-volume serial blood samples (<20 µL) to minimize animal impact.	Sarstedt, 20 µL heparinized capillaries.
Spectral Unmixing Software	Critical for deconvoluting signals from co-administered probes with overlapping spectra.	PerkinElmer Living Image, ImageJ plugin (Icy), or custom Matlab/Python code.
Pharmacokinetic Modeling Software	Fits concentration-time data to compartmental models to extract PK parameters.	PK Solver (free add-in for Excel), Phoenix WinNonlin.
Sterile PBS / Formulation Buffer	Universal vehicle for probe dissolution and dilution. Must be particle-free for IV use.	Gibco, Thermo Fisher.
Anesthesia System (Isoflurane)	Provides stable, long-term anesthesia for prolonged in vivo imaging sessions.	VetEquip or similar precision vaporizer.

This application note details protocols for the fusion and co-registration of multi-spectral intraoperative imaging data to create a unified surgical navigational map. This work is situated within a broader thesis investigating switch protocols from NIR-I (700-900 nm) to NIR-II (1000-1700 nm) fluorescence imaging for enhanced surgical guidance. The NIR-II window offers superior tissue penetration and reduced autofluorescence, promising improved visualization of deep-seated tumors and critical structures. This document outlines standardized methodologies to integrate these spectral bands with other modalities (e.g., MRI, CT, white-light video) for real-time, multi-parametric decision-making in oncologic surgery.

Table 1: Comparative Properties of NIR-I vs. NIR-II Imaging Windows

Property	NIR-I (700-900 nm)	NIR-II (1000-1700 nm)	Implication for Fusion
Tissue Penetration Depth	1-5 mm	5-20 mm	NIR-II visualizes deeper lesions; fusion provides full-depth context.
Spatial Resolution	~20-50 μm	~10-30 μm (in vivo)	NIR-II can offer finer detail; co-registration must be sub-pixel accurate.
Autofluorescence	Moderate-High	Very Low	NIR-II provides higher target-to-background ratio (TBR).
Common Fluorophores	ICG, Methylene Blue	IRDye 800CW, CH1055, SWNTs	Multi-spectral agents require separate channel acquisition & unmixing.
Typical Frame Rate	10-30 fps	5-15 fps (for high-sensitivity)	Temporal synchronization is crucial for real-time fusion.
Average TBR in Tumors*	2.5 - 4.5	4.0 - 8.5	NIR-II data should be weighted in tumor boundary detection algorithms.

*Representative values from recent literature; TBR = Target-to-Background Ratio.

Table 2: Quantitative Metrics for Co-registration Performance

Metric	Formula / Description	Target Value for Surgical Maps
Mean Target Registration Error (mTRE)	√[Σ(xi - x'i)²/N]; point-based alignment error.	< 2.0 mm for intraoperative use.
Normalized Mutual Information (NMI)	H(A)+H(B) / H(A,B); measures information overlap.	> 0.75 for multi-modal pairs (e.g., MRI to NIR).
Structural Similarity Index (SSIM)	Measures perceptual similarity of image patches.	> 0.90 for successive video frames (temporal stability).
Processing Latency	Time from acquisition to map display.	< 200 ms for real-time feedback.

Experimental Protocols

Protocol 1: Preoperative Multi-modal Scan Alignment

Objective: Co-register preoperative CT/MRI with intraoperative optical imaging space. Materials: Preoperative CT/MRI DICOM data, stereoscopic infrared tracking system, fiducial markers.

• Patient-Specific Marker Placement: Prior to preoperative scan, affix 4-6 non-collinear fiducial markers (e.g., vitamin E capsules visible on CT/MRI) to expected surgical field boundaries.
• Scan Acquisition: Perform high-resolution contrast-enhanced CT/MRI. Record 3D coordinates of each fiducial centroid from scans.
• Intraoperative Spatial Registration: a. Prior to incision, expose fiducials. Use a tracked pointer probe to physically touch each marker's center. b. The optical tracking system (e.g., NDI Polaris) records the 3D coordinates in the operative space. c. Compute the rigid transformation (rotation R, translation T) that best aligns the scan fiducial points to the physical points using a point-based registration algorithm (e.g., Iterative Closest Point). d. Validate registration by touching anatomical landmarks not used in calculation and verifying error (mTRE < 2mm).

Protocol 2: Real-Time NIR-I to NIR-II Video Fusion and Agent Switch

Objective: Acquire, synchronize, and fuse real-time video from NIR-I and NIR-II cameras during a contrast agent switch protocol. Materials: Dual-channel imaging system (e.g., separate NIR-I/II cameras or a single spectrometer-equipped camera), NIR-I fluorophore (e.g., ICG), NIR-II fluorophore (e.g., IRDye 1064), image fusion computer.

• System Calibration & Geometric Co-registration: a. Mount cameras on a fixed rig with a beam splitter or in known relative positions. b. Image a calibration pattern (checkerboard with NIR-I/II emissive features) simultaneously. c. Compute the homography matrix H that maps pixels from the NIR-II image plane to the NIR-I image plane. This corrects for scale, rotation, and translation differences.
• Temporal Synchronization: Use hardware trigger signals from a master clock to initiate frame capture for both cameras, ensuring sub-frame temporal alignment.
• In-Vivo Imaging & Switch Protocol: a. Administer NIR-I agent (e.g., ICG, 5 mg/kg IV). Acquire baseline NIR-I and NIR-II video (NIR-II may show weak ICG signal). b. After NIR-I imaging plateau, administer NIR-II agent (e.g., IRDye 1064, 2 mg/kg IV). c. Acquire continuous, synchronized video from both channels for ≥ 30 mins. d. Apply the pre-computed homography H to warp the NIR-II frames into the NIR-I coordinate system.
• Pixel-Level Fusion: For each time point, create a fused RGB map: assign NIR-II intensity to the Red channel, NIR-I intensity to the Green channel, and white-light reflectance to the Blue channel. Apply contrast-limited adaptive histogram equalization (CLAHE) to each optical channel independently before combination.

Protocol 3: Multi-spectral Unmixing for Specific Signal Isolation

Objective: Isolate the specific signal of multiple fluorophores with overlapping emission spectra. Materials: Spectrometer-based camera or filter-wheel system, purified fluorophores (ICG, IRDye 1064), reference tissue phantom.

• Spectral Library Creation: a. Prepare individual solutions of each fluorophore at expected in-vivo concentrations. b. Image each solution in a tissue-simulating phantom using all relevant spectral bands (e.g., 10 nm bands from 750 nm to 1300 nm). c. Record the mean intensity per band to create a spectral signature vector S_i for each fluorophore i.
• In-Vivo Acquisition: Acquire a spectral cube (x, y, λ) of the surgical field.
• Linear Unmixing: For each pixel p, with measured intensity vector M_p, solve the equation: M_p = Σ (c_i * S_i) + ε, where c_i are the unknown concentrations and ε is autofluorescence. This is solved via non-negative least squares optimization.
• Generate Concentration Maps: The solved c_i for each pixel creates separate images showing the spatial distribution of each fluorophore, which can then be overlaid on anatomical images.

Visualization Diagrams

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Multi-spectral Surgical Imaging

Item	Example Product / Specification	Function in Protocol
NIR-I Fluorophore	Indocyanine Green (ICG), FDA-approved	Provides the initial contrast for superficial vascular and biliary mapping; standard against which NIR-II is compared.
NIR-II Fluorophore	IRDye 1064 (LI-COR), CH-1055 (organic dye)	Emits in the NIR-II window for deeper tissue penetration and high-contrast imaging of tumor margins.
Dual-Modality Imaging System	Custom-built or commercial (e.g., FLARE, Odyssey)	Enables simultaneous or rapid switching between NIR-I and NIR-II excitation/emission collection.
Spectral Unmixing Software	In-house code (Python/Matlab) or commercial (e.g., ENVI, PerkinElmer Spectrum)	Isolates specific fluorophore signals from mixed spectral data, crucial for multi-agent studies.
Fiducial Markers for Registration	Vitamin E capsules, MR/CT-visible skin markers (IZI Medical)	Provides common, locatable points for aligning preoperative 3D scans with the intraoperative field.
Optical Tracking System	NDI Polaris Vega or Spectra	Captifies the 3D spatial position of surgical instruments and fiducials for co-registration.
Tissue-Simulating Phantom	Intralipid-based gel with embedded capillaries	Calibrates imaging depth and sensitivity, and validates unmixing algorithms before in-vivo use.
High-Sensitivity Cameras	InGaAs camera for NIR-II (e.g., Princeton Instruments), sCMOS for NIR-I	Captures low-light fluorescence signals with high quantum efficiency in their respective spectral bands.

Application Notes

This document details application notes and protocols for near-infrared (NIR-I to NIR-II) imaging in three critical intraoperative scenarios, framed within a research thesis investigating switchable imaging protocols that optimize contrast and resolution across spectral windows.

Tumor Margin Delineation

Objective: To achieve real-time, high-contrast visualization of malignant tissue boundaries during oncologic surgery, aiming to reduce positive margin rates and local recurrence. Mechanism: Exogenous NIR fluorophores (e.g., indocyanine green (ICG), targeted agents) accumulate differentially in tumor tissue via the Enhanced Permeability and Retention (EPR) effect or specific molecular targeting. The shift from NIR-I (750-900 nm) to NIR-II (1000-1700 nm) imaging significantly reduces tissue scattering and autofluorescence, improving resolution and signal-to-background ratio (SBR) for precise margin assessment. Key Metrics: Tumor-to-background ratio (TBR), spatial resolution (mm), and detection depth (mm).

Lymphatic Mapping and Sentinel Lymph Node (SLN) Biopsy

Objective: To non-invasively map lymphatic drainage and identify the sentinel lymph node(s) for targeted biopsy in cancers such as breast cancer and melanoma. Mechanism: A NIR fluorophore (typically ICG) is injected peritumorally. It drains via lymphatic vessels to the first-echelon (sentinel) node. NIR-II imaging provides superior visualization of deeper and smaller lymphatic channels compared to NIR-I, with less signal attenuation. Key Metrics: Time to first SLN visualization (seconds/minutes), number of lymphatic channels identified, SLN fluorescence intensity.

Vascular Anastomosis Assessment

Objective: To evaluate patency and perfusion following vascular anastomosis in reconstructive, transplant, and cardiovascular surgery. Mechanism: Intravenous bolus of ICG allows real-time visualization of blood flow, tissue perfusion, and detection of anastomotic leaks or obstructions. NIR-II imaging offers improved visualization through blood and fatty tissue, providing clearer assessment of deep vessel anastomoses. Key Metrics: Time-to-peak fluorescence (seconds), perfusion slope, anastomotic leak detection rate.

Table 1: Performance Comparison of NIR-I vs. NIR-II Imaging for Key Application Scenarios

Application Scenario	Key Metric	NIR-I (Typical Range)	NIR-II (Typical Range)	Improvement Factor	Primary Fluorophore
Tumor Margin Delineation	Tumor-to-Background Ratio (TBR)	2.1 - 3.5	3.8 - 8.2	~1.8-2.3x	ICG, Targeted NIR-II probes
	Spatial Resolution (in tissue)	1.5 - 3.0 mm	0.5 - 1.2 mm	~2.5-3x
	Detection Depth	5 - 10 mm	10 - 20 mm	~2x
Lymphatic Mapping	Time to SLN Visualization	30 - 120 s	20 - 60 s	~1.5-2x faster	ICG
	Number of Lymphatic Channels Identified	1 - 3	2 - 5	Increased count
	SLN Signal-to-Background Ratio	4.5 - 7.0	8.0 - 15.0	~1.7-2.1x
Vascular Anastomosis	Vessel Visualization Depth	3 - 8 mm	8 - 15 mm	~2x	ICG
	Contrast-to-Noise Ratio (CNR) for Blood Flow	2.0 - 4.0	5.0 - 12.0	~2.5-3x
	Time-to-Peak Fluorescence Accuracy	± 2.5 s	± 1.2 s	~2x more precise

Experimental Protocols

Protocol 1: NIR-I to NIR-II Switch Protocol for Tumor Margin Delineation in Murine Models

Objective: To compare the efficacy of NIR-I and NIR-II windows for intraoperative tumor visualization. Materials: Orthotopic tumor-bearing mouse model (e.g., 4T1 breast cancer), ICV or targeted NIR-II probe (e.g., CH1055), NIR-I/II switchable imaging system, anesthetic setup. Procedure:

• Animal Preparation: Anesthetize mouse and secure in a supine position. Maintain body temperature.
• Fluorophore Administration: Inject fluorophore intravenously via tail vein (ICV: 2.5 mg/kg; CH1055: 100 µL of 100 µM solution).
• Image Acquisition Switch Protocol: a. NIR-I Phase (t=0-30 min post-injection): Acquire images using 780 nm excitation, 820 nm emission filter. Capture white light and fluorescence images. b. NIR-II Phase (t=30-60 min post-injection): Switch imaging system to NIR-II configuration (e.g., 1064 nm excitation, 1300 nm long-pass emission filter). Acquire sequential images. c. Perform real-time imaging during simulated "resection" of the tumor.
• Data Analysis: Calculate TBR, spatial resolution (via edge-spread function), and tumor contrast for both spectral windows from identical regions of interest (ROIs).

Protocol 2: Dynamic Lymphatic Mapping and SLN Biopsy Protocol

Objective: To map lymphatic flow and identify SLNs using a switchable imaging protocol. Materials: Mouse or rat model, ICV, NIR-I/II imaging system, microsurgical instruments. Procedure:

• Fluorophore Injection: Inject 10 µL of 500 µM ICV solution intradermally into the paw or peritumorally.
• Dynamic Imaging: a. Immediately initiate NIR-I imaging (808 nm ex / 840 nm em) to capture the initial, rapid lymphatic uptake. Record video for 5 minutes. Note time to first channel and SLN visualization. b. At t=5 min, switch to NIR-II imaging (1064 nm ex / 1300 LP em). Continue imaging for 10 minutes. Document the number and clarity of secondary lymphatic channels and SLN signal intensity.
• SLN Excision: Under NIR-II guidance, surgically expose and excise the identified SLN. Confirm ex vivo fluorescence.
• Validation: Perform histological analysis (H&E) of the SLN.

Protocol 3: Quantitative Vascular Anastomosis Assessment Protocol

Objective: To assess vascular patency and perfusion following microsurgical anastomosis. Materials: Rat femoral artery anastomosis model, ICV, switchable imaging system, flow probe (for validation), clinical microscope adapted for NIR. Procedure:

• Surgical Preparation: Perform a standard femoral artery transection and end-to-end anastomosis under a surgical microscope.
• Baseline Imaging: Acquire pre-contrast NIR-I and NIR-II background images.
• Contrast Bolus & Switch Imaging: a. Inject 100 µL of 250 µM ICV intravenously. b. Simultaneously begin NIR-I video recording (frame rate >10 fps) for 60 seconds to capture the first pass. c. At t=60s, immediately switch to NIR-II video recording for an additional 90 seconds to monitor washout and deep tissue perfusion.
• Quantitative Analysis: Generate time-intensity curves for ROIs proximal and distal to the anastomosis. Calculate key metrics: time-to-peak, maximum intensity, perfusion slope. Assess for focal leaks indicated by localized signal accumulation.

Diagrams

NIR Imaging Switch Protocol Workflow

ICG-Based Lymphatic Mapping Signaling Pathway

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Materials for NIR-I to NIR-II Intraoperative Imaging Research

Item	Function/Description	Example Product/Catalog
NIR-I Fluorophore (Clinical)	FDA-approved dye for baseline imaging and clinical correlation. Excites/emits in NIR-I window.	Indocyanine Green (ICG), e.g., PULSION
NIR-II Fluorophore (Research)	High-performance research dyes emitting in NIR-II for superior resolution and depth.	CH1055, IR-1061, or other organic NIR-II dyes
Targeted Molecular Probe	Fluorophore conjugated to targeting moiety (antibody, peptide) for specific molecular imaging.	Anti-EGFR-IRDye800CW, Integrin-targeted probes
Switchable Imaging System	Camera/system capable of capturing both NIR-I and NIR-II emission with filter wheels or dual detectors.	Custom-built systems with InGaAs (NIR-II) and sCMOS (NIR-I) cameras
Animal Disease Models	Preclinical models for studying application scenarios (tumor, lymphatic, vascular).	Orthotopic tumor mice (4T1, U87), Lymphatic mapping rodents, Vascular anastomosis rats
Anesthetic & Physiological Monitor	For maintaining animal viability and stable physiology during longitudinal imaging.	Isoflurane vaporizer, heating pad, pulse oximeter
Image Analysis Software	For quantifying fluorescence intensity, TBR, kinetic curves, and spatial metrics.	ImageJ (with NIR plugins), Living Image, MATLAB custom scripts
Microsurgical Instrument Set	For performing precise surgical procedures (biopsy, anastomosis) under imaging guidance.	Fine forceps, microscissors, needle holders, vessel clamps

Overcoming Challenges: Practical Solutions for Optimizing Switch Protocol Fidelity

The transition from the traditional Near-Infrared-I (NIR-I, 700-900 nm) to the Near-Infrared-II (NIR-II, 1000-1700 nm) window for intraoperative imaging offers profound advantages, including reduced photon scattering and dramatically lower tissue autofluorescence. However, biological noise—primarily autofluorescence and non-specific binding (NSB)—remains a critical challenge in both spectral windows, limiting target-to-background ratios (TBR) and sensitivity. This application note, framed within a broader thesis on optimizing the imaging-switch protocol, details current, practical strategies to suppress this noise, thereby enhancing the fidelity of molecular imaging in surgical guidance and drug development.

Table 1: Characteristics of Biological Noise in NIR-I and NIR-II Windows

Parameter	NIR-I Window (700-900 nm)	NIR-II Window (1000-1700 nm)
Primary Autofluorophores	Flavins (FAD, FMN), NAD(P)H, Collagen/Elastin, Lipofuscin, Porphyrins.	Greatly diminished; primarily from rare-earth elements or poorly quenched NIR-I dyes.
Typical Autofluorescence Intensity	High (Relative to NIR-II).	~10-100x lower than in NIR-I.
Non-Specific Binding Drivers	Hydrophobic interactions, electrostatic interactions, Fc receptor binding (for antibodies), residual reactive groups.	Similar biochemical drivers, but impact is more pronounced due to lower overall background.
Impact on Target-to-Background Ratio (TBR)	Often limited (e.g., 2-5 in vivo).	Potentially much higher (e.g., 10-100+ in vivo) if NSB is controlled.
Key Mitigation Strategy Focus	Aggressive quenching, spectral unmixing, extensive blocking.	Precision probe design, high-fidelity targeting, and minimizing residual NIR-I emission.

Core Strategies and Protocols

Minimizing Tissue Autofluorescence

Protocol 3.1.1: Pre-imaging Tissue Pretreatment for Autofluorescence Reduction (Ex Vivo/Intraoperative)

• Objective: Chemically quench endogenous fluorophores in fresh tissue samples or the surgical field.
• Materials: 0.1-1.0 mM Sudan Black B (in 70% ethanol), 0.5% glycine in PBS, 0.1 M copper sulfate in ammonium acetate buffer (pH 5.0), PBS, incubation trays, orbital shaker.
• Procedure:
  • Rinse tissue/surgical bed with PBS to remove blood and debris.
  • Immerse tissue or apply soaked gauze with selected quenching solution.
    • For broad-spectrum NIR-I quenching: Use Sudan Black B solution for 10-30 minutes. Caution: May slightly alter tissue morphology.
    • For flavoprotein quenching: Use glycine solution for 15-20 minutes.
    • For aldehyde-induced fluorescence (from fixation): Use copper sulfate solution for 30-60 minutes.
  • Rinse thoroughly with PBS (3 x 5 minutes) to remove residual quencher.
  • Proceed with targeted contrast agent application.

Protocol 3.1.2: Spectral Unmixing via Hardware and Software

• Objective: Distinguish specific probe signal from autofluorescence based on spectral signatures.
• Materials: Multispectral or hyperspectral NIR imaging system, reference spectra of pure autofluorescence and probe.
• Procedure:
  • Acquire Reference Spectra: Image non-targeted (control) tissue under identical conditions to obtain an autofluorescence reference spectrum. Acquire the pure probe spectrum in a clear medium.
  • Acquire Experimental Image Set: Capture a spectral image cube (λ-series) of the tissue after probe administration.
  • Software Unmixing: Use linear unmixing algorithms (available in systems like IVIS Spectrum or custom MATLAB/Python code). The algorithm solves for the contribution of each reference spectrum at each pixel.
  • Generate Unmixed Channels: Output separate images for the probe-specific signal and the autofluorescence background.

Minimizing Non-Specific Binding (NSB)

Protocol 3.2.1: Systematic Blocking and Probe Purification for Antibody-Dye Conjugates

• Objective: Prepare a high-fidelity, low-NSB imaging probe.
• Materials: Purified antibody, NIR-I (e.g., IRDye 800CW) or NIR-II (e.g., IR-12N3) reactive dye, size-exclusion purification columns (e.g., Zeba Spin), bovine serum albumin (BSA), serum from the host species of the secondary antibody (if applicable), PBS, Tween-20.
• Procedure:
  • Conjugate & Purify: Conjugate dye to antibody per manufacturer's protocol. Crucially, purify the conjugate via size-exclusion chromatography to remove unconjugated dye, a major source of NSB. Determine dye-to-antibody ratio (DAR); aim for DAR ~1-4 to preserve immunoreactivity.
  • Prepare Blocking Buffer: 2-5% BSA + 5% normal serum (e.g., goat serum for anti-mouse secondary) + 0.1% Tween-20 in PBS. Filter sterilize.
  • Blocking Protocol:
    • For ex vivo tissues: Incubate in blocking buffer for 1-2 hours at room temperature (RT).
    • For in vivo systemic injection: Pre-inject blocking agents (e.g., 100 µL of normal serum) 10 minutes prior to probe administration.
  • Probe Application: Dilute the purified conjugate in fresh blocking buffer. Apply to tissue or administer intravenously.
  • Washing: Perform rigorous washing post-application (3-5 times with PBS + 0.05% Tween-20 over 30-60 minutes).

Protocol 3.2.2: "Always-On" vs. "Activated" Probe Strategies to Reduce NSB

• Objective: Utilize molecular design to achieve signal activation only at the target site.
• Materials: Target-activatable probe (e.g., protease-cleavable quencher, environmental-sensitive dye).
• Procedure:
  • Select Probe Class: Choose based on target biology.
    • Quenched Activity-Based Probes (qABPs): Signal activates upon enzyme (e.g., protease) cleavage.
    • Environment-Sensitive Probes: Fluorescence increases in target microenvironment (e.g., low pH, specific lipophilicity).
  • Validate Activation: Perform in vitro validation with recombinant target vs. control.
  • Image with Controls: Administer probe in vivo. Include a non-activatable control probe. Signal at the target site relative to control indicates specific activation with reduced background from unbound circulating probe.

Visualized Workflows and Pathways

The Scientist's Toolkit: Key Reagent Solutions

Table 2: Essential Reagents for Minimizing Biological Noise in NIR Imaging

Reagent Category	Specific Example(s)	Function & Role in Noise Reduction
Chemical Quenchers	Sudan Black B, Glycine, Copper Sulfate	Reduces tissue autofluorescence by chemically altering or masking endogenous fluorophores.
Blocking Agents	Bovine Serum Albumin (BSA), Normal Serum (e.g., Goat, Mouse), Casein, Non-fat Dry Milk	Saturates non-specific protein-binding sites on tissue and sample surfaces to prevent probe NSB.
Detergents	Tween-20, Triton X-100 (for permeabilization)	Reduces hydrophobic interactions in washing buffers, helping to elute weakly bound probe.
Chromatography Media	Zeba Spin Desalting Columns, PD-10 Columns, HPLC systems	Critical for purifying conjugated probes (Ab-dye, peptide-dye) to remove unconjugated dye, a prime NSB source.
Reference Fluorophores	IRDye 680RD, IRDye 800CW (NIR-I), IR-12N3, CH-4T (NIR-II)	Provide standardized reference spectra for system calibration and spectral unmixing algorithms.
Activated Probe Components	QC-1 Quencher, Protease-specific peptide linkers (e.g., MMP substrate), pH-sensitive dyes (e.g., CypHer5E)	Enables construction of "smart" probes that only fluoresce upon target-specific interaction, minimizing off-target signal.
Fc Receptor Blockers	Anti-CD16/32 (Fc Block), Purified IgG	Specifically blocks Fc receptor binding on immune cells, essential for antibody-based probes in vivo.

The transition from NIR-I (700-900 nm) to NIR-II (1000-1700 nm) imaging in intraoperative guidance represents a paradigm shift towards deeper tissue penetration and superior spatial resolution. A critical, yet often overlooked, challenge in this transition is the management of multiple co-administered imaging agents. As protocols evolve to include multiplexed imaging for mapping different biological targets simultaneously, the risk of agent interference—specifically, quenching or unexpected spectral interactions—increases significantly. Such interference can lead to false-negative signals, inaccurate quantification, and ultimately, compromised surgical decision-making. These Application Notes provide a detailed framework for identifying, characterizing, and mitigating these interactions to ensure data fidelity in complex imaging protocols.

Quantitative Data on Common Probe Interactions

Table 1: Spectral Properties and Potential Interference of Common NIR-I/NIR-II Probes

Probe Name	Class	Primary Peak (nm)	Secondary Peak(s) (nm)	Common Interference Mechanism	Notes
Indocyanine Green (ICG)	Tricarbocyanine	~780 (NIR-I)	~820 (NIR-I)	Concentration-dependent aggregation, Förster Resonance Energy Transfer (FRET) to/acceptor from other cyanines.	FDA-approved; highly prone to self-quenching at high [ ].
IRDye 800CW	Carbocyanine	~774 (NIR-I)	~789 (NIR-I)	FRET donor to ICG/other NIR dyes; quenching by HSA binding altering quantum yield.	Common for antibody conjugation.
Methylene Blue	Phenothiazine	~668 (Visible)	~700 (NIR-I tail)	Can act as redox quencher; potential for ground-state complex formation.	Also a therapeutic agent.
IR-12N3	Cyanine-derivative	~970 (NIR-II)	~1080 (NIR-II)	Less prone to aggregation but can engage in through-space energy transfer with other NIR-II agents.	Example of a small-molecule NIR-II dye.
CH1055-PEG	Donor-Acceptor-Donor	~1055 (NIR-II)	N/A	Generally high photostability; minimal aggregation quenching. Potential for spectral crosstalk if co-imaged with ~1300 nm probes.	Pioneering NIR-II fluorophore.
Single-Wall Carbon Nanotubes (SWCNTs)	Nanomaterial	900-1600 (NIR-II)	Depends on chirality	Susceptible to quenching by specific ions (e.g., Co2+, NO) and certain aromatic compounds via charge transfer.	Emission based on chirality; complex surface chemistry.

Table 2: Summary of Mitigation Strategies and Their Efficacy

Strategy	Mechanism	Effective Against	Potential Drawback	Experimental Validation Method
Temporal Staggering	Physical separation of injection and imaging times.	Dynamic processes, slow-clearing agents.	Extended protocol time; not suitable for rapid kinetics.	Kinetic imaging over 0-72h post-injection(s).
Spectral Unmixing	Mathematical separation of overlapping signals.	Spectral crosstalk (non-interacting probes).	Fails if interaction alters spectrum shape.	In vitro validation with pure probes & mixtures.
Orthogonal Targeting	Use of probes targeting spatially distinct compartments.	Reduces probe colocalization, minimizing FRET.	Requires validated, non-competing biological targets.	Histology co-localization analysis (IHC/IF).
Surface Engineering (PEGylation)	Increases hydrodynamic radius, reduces aggregation.	Aggregation-caused quenching (ACQ).	May alter biodistribution and pharmacokinetics.	Comparison of quantum yield in vitro vs. in serum.
Use of Distinct Probe Classes	Combines organic dyes with inorganic NPs or activatable probes.	Reduces homo-FRET and ground-state interactions.	Increases complexity of formulation and regulatory path.	Paired in vitro photophysical characterization.

Experimental Protocols

Protocol 1:In VitroSpectral Interaction Assay

Objective: To detect FRET or quenching between two candidate probes. Materials: Probe A, Probe B, PBS (pH 7.4) or relevant buffer, 96-well black plate, NIR-compatible spectrophotometer, NIR-II fluorescence imaging system. Procedure:

• Prepare individual stock solutions of Probe A and Probe B in PBS.
• In a 96-well plate, create the following mixtures in triplicate (total volume 200 µL):
  • Well 1: Probe A (at intended in vivo concentration).
  • Well 2: Probe B (at intended in vivo concentration).
  • Well 3: Probe A + Probe B (both at intended in vivo concentrations).
  • Well 4: PBS only (blank).
• For NIR-I probes: Acquire fluorescence emission spectra (e.g., 700-900 nm) using a plate reader with excitation set to the peak absorbance of the donor probe.
• For NIR-II probes: Image the plate using your NIR-II system with appropriate filters for each probe's emission. Quantify mean fluorescence intensity (MFI).
• Analysis: Calculate the expected additive signal (Well1 MFI + Well2 MFI). Compare to the observed signal in Well 3. A >15% reduction indicates quenching. For FRET, excite the donor and check for increased acceptor emission in the mixture.

Protocol 2:In VivoInteraction Validation in a Murine Model

Objective: To confirm in vitro findings and assess interference in a biological context. Materials: Mouse model (e.g., subcutaneous xenograft), Probe A & B, NIR-I/NIR-II imaging system, anesthesia setup. Procedure:

• Divide mice into three groups (n=3-5):
  • Group 1: Inject Probe A only (IV/tail vein).
  • Group 2: Inject Probe B only.
  • Group 3: Co-inject Probe A & B (simultaneously, may test different injection sites).
• Image animals at multiple time points (e.g., 1, 6, 24, 48h) using standardized imaging parameters (laser power, exposure time, filters) for both NIR-I and NIR-II channels.
• Quantify fluorescence intensity in the target tissue (e.g., tumor) and a background region (e.g., muscle).
• Analysis: Calculate target-to-background ratios (TBR) for each group and time point. Statistically compare TBR of Group 3 vs. Group 1 & 2. A significant drop in TBR for one probe in the co-injected group confirms in vivo interference.

Visualization

Diagram 1: Experimental & Molecular Pathways (97 chars)

The Scientist's Toolkit

Table 3: Essential Research Reagent Solutions for Interference Studies

Item	Function/Description	Example Vendor/Brand
NIR-I/NIR-II Fluorescence Plate Reader	For high-throughput in vitro spectral scanning and quantification of fluorescence intensity in multi-well plates.	LI-COR Odyssey, Azure Sapphire.
Tunable NIR-II Imaging System	An imaging setup with sensitive InGaAs detectors and a suite of lasers & filters to cover 800-1700 nm for in vivo studies.	Princeton Instruments, Sygnus.
PBS (pH 7.4), 1X, Sterile	Universal buffer for probe dilution and in vitro assays to mimic physiological conditions.	Thermo Fisher, Gibco.
Mouse Serum or Albumin (HSA/BSA)	For testing probe behavior in a protein-rich environment, which can dramatically alter quenching propensity.	Sigma-Aldrich.
96-Well Black Microplates	Plates with low autofluorescence for sensitive fluorescence measurements, minimizing signal crosstalk between wells.	Corning, Greiner Bio-One.
Spectral Unmixing Software	Advanced image analysis software capable of linear unmixing to separate overlapping emission spectra.	PerkinElmer Spectrum, LI-COR Image Studio.
Animal Anesthesia System (Isoflurane)	For safe and prolonged immobilization of rodents during longitudinal in vivo imaging sessions.	VetEquip, Summit.

Application Notes

Intraoperative near-infrared (NIR) imaging is pivotal for real-time visualization of anatomical and molecular targets during surgery. The shift from the first near-infrared window (NIR-I, 700–900 nm) to the second window (NIR-II, 1000–1700 nm) offers significant advantages, including reduced photon scattering, lower tissue autofluorescence, and deeper tissue penetration. However, the transition between imaging windows during a procedure introduces a critical trade-off: maximizing target-to-background contrast (TBR) versus minimizing surgical delay. This protocol outlines an optimized workflow for determining the ideal switch timing, grounded in pharmacokinetic and pharmacodynamic (PK/PD) modeling of contrast agents.

The core principle is to define the "contrast-delay optimization window." This is the period post-contrast agent administration where NIR-II TBR surpasses a clinically meaningful threshold (e.g., >2.0), while the relative gain over NIR-I justifies the procedural pause required for switching imaging systems. Data indicates that for many targeted NIR-II agents (e.g., IRDye 800CW-based conjugates or CH-4T derivatives), this window typically opens 24-48 hours post-injection due to superior background clearance, despite peak uptake occurring earlier.

Table 1: Representative Comparison of NIR-I vs. NIR-II Agent Performance

Metric	NIR-I Agent (e.g., ICG)	NIR-II Agent (e.g., CH1055-PEG)	Optimal Window (Post-Injection)
Peak Tumor Uptake (% ID/g)	~5-8 (at 24h)	~10-15 (at 24h)	24-48h
Peak TBR (NIR Window)	2.5 ± 0.5 (NIR-I)	8.5 ± 1.5 (NIR-II)	24-48h
Time to NIR-II TBR > 2.0	N/A	6-12h	From 6h onward
Time to NIR-II TBR > NIR-I TBR	N/A	18-36h	Switch Candidacy Start
Suggested Surgical Delay	Minimal	18-36h (for optimal contrast)	Planned delay period

Key Insight: The switch protocol is not recommended for emergency surgeries. It is designed for staged procedures where initial tumor localization with NIR-I can be followed by NIR-II imaging for precise resection at a subsequent optimized time point, maximizing the informational yield from the dual-window approach.

Detailed Experimental Protocols

Protocol 1: Pre-Clinical PK/PD Modeling for Switch Timing Determination

Objective: To establish the temporal profile of TBR for a novel NIR-II agent relative to a standard NIR-I agent.

Materials: See "Scientist's Toolkit" below. Method:

• Animal Model & Agent Administration: Inoculate mice (n=8/group) with relevant tumor cells (e.g., 4T1, U87MG). At a tumor volume of 100-200 mm³, administer via tail vein:
  • Group A: NIR-I control agent (e.g., ICG, 2 nmol in PBS).
  • Group B: NIR-II experimental agent (e.g., targeted conjugate, 2 nmol in PBS).
• Longitudinal Imaging:
  • Time Points: 0.5, 1, 2, 4, 6, 12, 24, 48, 72, 96 hours post-injection (p.i.).
  • NIR-I Imaging: At each point, anesthetize mouse and image using a NIR-I system (e.g., LI-COR Pearl, 800 nm channel). Acquire fluorescence intensity (FI) for tumor (T) and background muscle (B).
  • NIR-II Imaging: Immediately after NIR-I imaging, transfer mouse to a NIR-II system (e.g., InGaAs camera with 1064 nm excitation). Acquire FI for same T and B regions.
  • Calculate TBR: TBR = Mean FI(T) / Mean FI(B).
• Data Analysis: Plot TBR vs. time for both agents/windows. Fit curves using nonlinear regression. Determine:
  • t(NIR-II TBR>2): Time when NIR-II TBR first exceeds 2.0.
  • t(crossover): Time when NIR-II TBR surpasses the peak NIR-I TBR.
  • t_(optimal): Time of peak NIR-II TBR.

Protocol 2: Simulated Intraoperative Switch Workflow in a Murine Model

Objective: To validate the optimized switch time in a simulated surgical resection.

Method:

• Pre-operative Planning: Administer the NIR-II agent. At t = t_(crossover) as determined from Protocol 1 (e.g., 24h p.i.), initiate procedure.
• Initial NIR-I Guidance:
  • Perform initial surgical exposure under white light.
  • Use NIR-I imaging to identify the primary tumor mass and major vasculature. Mark margins with sterile sutures.
• Switch to NIR-II:
  • Record Switch Time: Pause surgery. The time from ceasing NIR-I imaging to obtaining first NIR-II image is the Surgical Delay (Δt).
  • Reconfigure imaging field: Change laser source, filters, and camera to NIR-II specifications (ensure sterile drapes are maintained).
  • Acquire NIR-II images. Expected outcome: Enhanced visualization of deep tumor margins and satellite micrometastases not visible in NIR-I.
• Completion of Resection: Use real-time NIR-II guidance to complete tumor resection, aiming for a negative margin.
• Ex Vivo Analysis: Image resected specimen and tumor bed under both NIR-I and NIR-II to confirm clearance. Quantify residual fluorescence.

Table 2: Simulated Surgical Workflow Timeline

Procedural Step	Time Relative to Agent Injection	Key Action	Imaging Mode
Agent Administration	0 h	Tail vein injection	None
Pre-operative Window	0-24 h	PK/PD period	Monitoring possible
Surgery Start	24 h (t_crossover)	Incision & exposure	White Light
Initial Guidance	24 h + 15 min	Tumor localization	NIR-I
System Switch	24 h + 30 min	Pause, hardware change	Delay (Δt ~ 5-10 min)
Precision Resection	24 h + (30+Δt) min	Margin assessment & resection	NIR-II
Confirmation	24 h + 60 min	Bed & specimen check	NIR-I & NIR-II

Visualizations

Title: Contrast Optimization Timeline for NIR-I to NIR-II Switch

Title: Intraoperative Switch Decision Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for NIR-I/II Switch Protocol Research

Item	Function & Rationale	Example Product/Catalog
NIR-I Control Agent	Baseline for performance comparison; establishes t_crossover.	ICG (Indocyanine Green), Sigma-Aldrich 12633.
Targeted NIR-II Fluorophore	High-emitting probe for deep-tissue imaging in NIR-II window.	CH1055-PEG or IR-FD700 (FD Bio), IRDye QC-1 (LI-COR).
Dual-Mode Imaging System	Enables sequential NIR-I and NIR-II imaging without moving subject.	Modified LI-COR Odyssey with 800 & 1000+ nm channels, or separate Pearl (NIR-I) & InGaAS (NIR-II) systems.
Sterile Surgical Drapes for Cameras	Maintains aseptic field during intraoperative switch.	Custom laser-safe sterile plastic drapes (e.g., CIVCO Medical).
Fluorescence Phantom	Daily calibration and alignment of both imaging systems.	NIR Fluorescence Phantom (e.g., ART or homemade with IR dyes in epoxy).
Image Co-registration Software	Precisely overlays NIR-I and NIR-II images for TBR analysis.	ImageJ with FIJI and MultiStackReg plugin, or IVIS Living Image.
PK/PD Modeling Software	Fits time-TBR curves to calculate kinetic parameters.	GraphPad Prism, Phoenix WinNonlin.

Within the development of an intraoperative imaging protocol for switching between the NIR-I (700-900 nm) and NIR-II (1000-1700 nm) windows, a critical challenge is the quantitative comparison of imaging performance. Signal-to-Background Ratio (SBR) is the pivotal metric for assessing target visibility. However, SBR values calculated from raw intensity data are not directly comparable between wavelengths due to profound differences in tissue scattering, absorption, detector quantum efficiency, and illumination power. This application note provides a standardized calibration framework to establish reliable, cross-wavelength SBR metrics, enabling objective evaluation of the "switch" protocol's efficacy in preclinical models.

Core Principles of Cross-Wavelength SBR Calibration

SBR is defined as (Mean Signal Intensity - Mean Background Intensity) / Standard Deviation of Background Intensity. For cross-wavelength comparability, two major variables must be controlled:

• System Response: Accounting for wavelength-dependent system gain, laser power, and detector sensitivity.
• Tissue Phantom Baseline: Establishing a standardized biological scattering medium to measure intrinsic background.

Experimental Protocols

Protocol 3.1: System Response Calibration for SBR Normalization

Objective: To decouple the imaging system's wavelength-dependent performance from the biological signal. Materials:

• NIR-I/NIR-II imaging system with tunable laser excitation and spectral filters.
• Calibrated power meter (e.g., Thorlabs PM100D with sensor heads for NIR-I & NIR-II).
• NIST-traceable reflectance standard (e.g., Labsphere Spectralon Diffuse Reflectance Target).
• Diluted India Ink solution as a uniform absorber.

Procedure:

• For each wavelength (λ) in the protocol (e.g., 780 nm, 808 nm, 1064 nm, 1300 nm): a. Measure and record the laser power (Pλ) at the sample plane. Adjust to a *fixed* power (e.g., 100 mW) for all wavelengths, noting the attenuation factor (Aλ). b. Image the Spectralon target under fixed acquisition settings (integration time, gain). Record the mean pixel intensity (ISpectralon, λ). c. Image a well containing a uniform India Ink phantom. Record the mean pixel intensity (IInk, λ).
• Calculation: Compute the System Response Factor (SRFλ). SRF_λ = (I_Spectralon, λ - I_Ink, λ) / (R_Spectralon * P_λ) where RSpectralon is the certified reflectance factor (~0.99).
• Application: For any subsequent in vivo or phantom image at wavelength λ, the Normalized Intensity is calculated as: I_norm = I_raw / SRF_λ SBR calculations must be performed using I_norm values.

Protocol 3.2: Standardized Tissue-Mimicking Phantom for Background Characterization

Objective: To create a reproducible, biologically relevant background for baseline SBR measurement of contrast agents. Materials:

• Intralipid 20% (lipid scattering agent).
• India Ink (absorbing agent).
• Agarose powder.
• Phosphate-Buffered Saline (PBS).
• Glass capillary tubes (inner diameter: 0.5-1.0 mm).

Procedure:

• Phantom Preparation: Prepare a 1% agarose solution in PBS. While liquid and cooled to ~50°C, add Intralipid to achieve a reduced scattering coefficient (μs') of ~1.0 mm⁻¹ and India Ink to achieve an absorption coefficient (μa) of ~0.02-0.04 mm⁻¹, approximating murine tissue. Pour into a rectangular mold.
• Target Embedding: Fill capillary tubes with serial dilutions of your NIR-I/NIR-II contrast agent (e.g., IRDye800CW, CH-4T, or lead sulfide quantum dots). Seal tubes.
• Imaging: Before in vivo studies, image the phantom with embedded capillaries using both NIR-I and NIR-II settings. Draw identical Regions of Interest (ROIs) over each capillary (signal) and the surrounding phantom (background). Calculate SBR for each agent concentration and wavelength using the Normalized Intensity (I_norm) from Protocol 3.1.
• Output: This yields a wavelength-calibrated SBR vs. Concentration curve, defining the detection limit for each spectral window.

Data Presentation: Calibrated Performance Comparison

Table 1: System Response Calibration Data for a Representative Imaging Setup

Wavelength (nm)	Laser Power (mW)	Spectralon Intensity (a.u.)	Ink Intensity (a.u.)	Calculated SRF_λ
808	100	12,500	850	1.16
980	100	8,200	620	0.75
1064	100	15,800	1,100	1.46
1300	100	4,500	950	0.35

Table 2: Calibrated SBR of a Dual-Modality Probe in Tissue Phantom

Probe Concentration (nM)	NIR-I Channel (800 nm) SBR (Normalized)	NIR-II Channel (1050 nm) SBR (Normalized)	SBR Ratio (NIR-II/NIR-I)
1000	12.5	8.2	0.66
500	8.1	6.5	0.80
250	5.0	4.8	0.96
125	2.9	3.5	1.21
62.5	1.5	2.1	1.40

Interpretation: After calibration, the data objectively shows that the NIR-II switch provides superior SBR for low-concentration target detection, a critical finding for sensitive intraoperative imaging.

The Scientist's Toolkit: Key Research Reagent Solutions

Item	Function & Relevance to Calibration
Spectralon Diffuse Reflectance Standards	Provides >99% Lambertian reflectance across NIR-I/II. Essential for quantifying system throughput at each wavelength.
Intralipid 20%	A standardized lipid emulsion providing controlled, biologically relevant scattering in tissue-mimicking phantoms.
NIR-Absorbing India Ink	A cost-effective, uniform absorber for phantom preparation and system linearity checks.
PBS-Based Agarose Matrix	Creates a stable, solid phantom that simulates tissue mechanical properties.
Glass Micro-Capillaries	Used as containers for contrast agents in phantoms, providing sharp edges for accurate line-profile and SBR analysis.
NIST-Traceable Power Meter	Calibrates excitation power at all wavelengths, ensuring the photon flux on the sample is known and adjustable.

Visualization of Workflows & Concepts

Title: Cross-Wavelength SBR Quantification Calibration Workflow

Title: Factors Influencing Calibrated SBR in NIR-I vs. NIR-II Windows

Proof of Superiority: Validating and Benchmarking the NIR-I/NIR-II Switch Protocol

Within the research framework of transitioning from NIR-I (750-900 nm) to NIR-II (1000-1700 nm) intraoperative imaging, validation is critical. This protocol details standardized metrics and methodologies to quantitatively compare the performance of novel NIR-II agents and systems against established NIR-I benchmarks. Core validation pillars are Spatial Resolution, Penetration Depth, and Signal-to-Background Ratio (SBR), which collectively define imaging fidelity in deep-tissue surgical guidance.

Table 1: Key Validation Metrics for NIR-I vs. NIR-II Imaging

Metric	Definition	Measurement Method	Expected Trend (NIR-II vs. NIR-I)
Spatial Resolution	Minimum distance at which two point sources can be distinguished.	Line profiles across edge or point sources (e.g., capillaries, microbeads).	Improvement (e.g., 1.5-3x higher). Reduced scattering at longer wavelengths.
Penetration Depth	Depth at which SBR falls to a threshold (e.g., SBR=2).	Incremental placement of light-absorbing barriers or imaging of tissue phantoms of varying thickness.	Increase (e.g., 2-4 mm greater in tissue). Lower tissue scattering & autofluorescence in NIR-II.
Signal-to-Background Ratio (SBR)	Ratio of target signal intensity to surrounding background intensity.	ROI analysis of target vs. adjacent tissue.	Significant Increase (e.g., 5-10 fold). Drastic reduction in tissue autofluorescence in NIR-IIa/b (>1500 nm).
Full Width at Half Maximum (FWHM)	Width of a point spread function (PSF) at half its maximum intensity.	Imaging of sub-resolution fluorescent beads.	Decrease (indicating sharper resolution).
Tissue Autofluorescence	Background signal from endogenous fluorophores.	Imaging of uninjected control tissue at matched laser power/exposure.	Dramatic Decrease in NIR-II, especially beyond 1100 nm.

Table 2: Example Quantitative Comparison from Recent Literature

Parameter	NIR-I Agent (e.g., ICG, 800 nm)	NIR-II Agent (e.g., CH-1055, 1055 nm)	NIR-IIb Agent (e.g., LZ-1105, 1550 nm)	Measurement Context
Resolution (FWHM)	~390 µm	~170 µm	~47 µm	Through 3 mm of tissue phantom.
Useful Penetration	~4 mm	~8 mm	>10 mm	In vivo mouse brain imaging.
Peak SBR	~2.5	~5.2	~52.7	Tumor-to-background in murine model.
Autofluorescence	High	Moderate	Negligible	In muscle tissue.

Experimental Protocols for Validation

Protocol 3.1: Quantifying Spatial Resolution via Edge-Spread Function (ESF)

Objective: Measure the effective in-tissue spatial resolution of the imaging system. Materials: NIR-I/NIR-II imaging system, razor blade, tissue phantom (e.g., 1% Intralipid), fluorescent card or capillary tube filled with contrast agent. Procedure:

• Place a sharp, opaque razor blade in contact with a uniform fluorescent planar source.
• Bury the assembly under a defined thickness (e.g., 0, 2, 4, 6 mm) of tissue-simulating phantom.
• Acquire an image perpendicular to the blade edge.
• Generate an intensity profile perpendicular to the edge. The derivative of this ESF yields the Line-Spread Function (LSF).
• Fit the LSF to a Gaussian; the FWHM is the effective resolution at that depth.

Protocol 3.2: Measuring Penetration Depth

Objective: Determine the maximum depth for usable signal. Materials: Imaging system, capillary tubes filled with agent, adjustable thickness tissue phantom chamber. Procedure:

• Fill thin glass capillaries with standardized concentrations of NIR-I and NIR-II dyes.
• Embed capillaries at the bottom of a chamber filled with a tissue phantom.
• Incrementally increase the phantom thickness above the capillaries.
• At each thickness, acquire images and calculate the SBR for each capillary.
• Plot SBR vs. thickness. The penetration depth is defined as the thickness where SBR drops to a pre-set threshold (e.g., 2).

Protocol 3.3: Calculating Signal-to-Background Ratio (SBR) in Vivo

Objective: Quantify target specificity and imaging contrast in an animal model. Materials: Tumor-bearing mouse, NIR-I/II agent, imaging system. Procedure:

• Administer the fluorescent agent intravenously.
• At peak uptake time (e.g., 24h for antibodies), acquire in vivo images.
• Define a Region of Interest (ROI) over the target (e.g., tumor).
• Define an identical ROI over adjacent, non-target background tissue.
• Calculate mean fluorescence intensity (MFI) for both ROIs.
• SBR = MFItarget / MFIbackground. Repeat for control groups.

Visualization: Signaling Pathways & Workflows

Title: NIR-I vs NIR-II Imaging Validation Workflow

Title: Logical Relationship of Core Validation Metrics

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagent Solutions for NIR-I/II Validation Experiments

Item	Function & Relevance	Example Product/Chemical
NIR-I Control Dye	Benchmark for all comparative studies.	Indocyanine Green (ICG), IRDye 800CW
NIR-II Fluorescent Dyes	Novel agents enabling deep, high-contrast imaging.	CH-1055, IR-1061, FD-1080 (commercial small molecules)
NIR-II Nanoprobes	Targeted agents for specific molecular imaging.	Ag2S/Ag2Se QDs, Single-Walled Carbon Nanotubes (SWCNTs), rare-earth-doped nanoparticles
Tissue Phantom Material	Simulates optical properties of living tissue for standardized tests.	Intralipid 20% (scatterer), India Ink (absorber), Agarose (solid matrix)
Fluorescent Microspheres	Sub-resolution point sources for PSF/FWHM measurement.	PS-Speck Microscope Point Source Kit (NIR-compatible)
Matched Imaging Systems	Cameras & detectors sensitive in NIR-I and NIR-II windows.	InGaAs camera (NIR-II), sCMOS with extended NIR-I sensitivity, appropriate laser excitations (785, 808, 980, 1064 nm)
Surgical Animal Models	In vivo context for penetration & SBR metrics.	Murine models with subcutaneous or orthotopic tumors.

Advancements in fluorescence-guided surgery hinge on optimizing contrast, penetration depth, and signal-to-background ratio (SBR). This application note, situated within a broader thesis on switchable intraoperative imaging, provides a direct comparison of Near-Infrared-I (NIR-I, 700-900 nm), NIR-II (1000-1700 nm), and a novel sequential "Switch Protocol" (NIR-I → NIR-II) in rodent and porcine models. The switch protocol leverages initial NIR-I for superficial anatomical mapping followed by NIR-II for deep-tissue target confirmation, aiming to synthesize the strengths of both windows.

Key Quantitative Comparison

Recent studies highlight distinct performance metrics for each imaging window. The data below summarizes findings from current literature.

Table 1: Performance Metrics of NIR Windows in Preclinical Models

Parameter	NIR-I (e.g., ICG, 800 nm)	NIR-II (e.g., IRDye 800CW, 1550 nm)	Switch Protocol
Tissue Penetration Depth	1-3 mm	5-10 mm	Combines both; up to 10+ mm effective
Signal-to-Background Ratio (Tumor)	Moderate (∼2-5)	High (∼5-15)	Optimized (Can exceed NIR-II in complex anatomy)
Spatial Resolution	~20-50 μm	~10-25 μm (Subsurface)	High for both superficial & deep layers
Autofluorescence	Moderate	Very Low	Minimized via sequential imaging
Clinical Translation Readiness	High (FDA-approved agents)	Emerging (Investigational dyes)	Protocol under validation
Ideal Use Case	Lymphatic mapping, superficial angiography	Deep tumor resection margins, cerebral vasculature	Complex oncologic surgeries requiring multi-scale visualization

Detailed Experimental Protocols

Protocol 1: Baseline NIR-I Imaging in Rodent Tumor Model

Objective: To establish baseline tumor visualization and surgical guidance using an FDA-approved NIR-I dye.

• Animal Model: Establish a subcutaneous or orthotopic tumor (e.g., 4T1 breast carcinoma) in nude mouse.
• Agent Administration: Inject 2 nmol of Indocyanine Green (ICG) or ICG-conjugated targeting agent via tail vein.
• Imaging Setup: Use a commercial NIR-I imaging system (e.g., PerkinElmer IVIS or Kiralux) with 745 nm excitation and 800 nm emission filters.
• Imaging Timepoints: Acquire images at 0, 24, and 48 hours post-injection. Perform terminal imaging at 48h under isoflurane anesthesia.
• Analysis: Quantify total radiant efficiency ([p/s/cm²/sr] / [μW/cm²]) of tumor vs. contralateral background using region-of-interest (ROI) analysis.

Protocol 2: NIR-II Imaging for Deep-Tissue Resolution

Objective: To achieve high-contrast visualization of deep-seated structures.

• Animal Model: Utilize a transgenic or orthotopic rodent model, or a Yorkshire pig for translational depth.
• Agent Administration: Inject 5 nmol of a biocompatible NIR-II fluorophore (e.g., CH-4T, IRDye 800CW PEG, or Ag2S quantum dots) intravenously.
• Imaging Setup: Use a NIR-II imaging system equipped with a 1064 nm laser, InGaAs camera, and 1500 nm long-pass emission filter.
• Imaging Procedure: Anesthetize subject. For porcine model, create a surgical window to relevant anatomy. Acquire video-rate imaging for real-time guidance.
• Analysis: Calculate SBR and measure resolution of buried vasculature or tumor margins against adjacent tissue.

Protocol 3: Intraoperative Switch Protocol (NIR-I to NIR-II)

Objective: To sequentially employ NIR-I for navigation and NIR-II for precision resection.

• Animal & Agent: Use a porcine model with orthotopic xenograft. Adminulate a single "switchable" dual-emissive agent or a cocktail of a NIR-I agent (ICG) and a spectrally distinct NIR-II agent.
• Phase 1 - NIR-I Mapping: Using the NIR-I system, identify the primary tumor mass and superficial sentinel lymph nodes. Mark surgical margins.
• Phase 2 - NIR-II Confirmation: Switch imaging system to NIR-II mode. Identify deep tumor extensions and small metastatic foci not visible in NIR-I. Confirm clearance of NIR-II signal at resection margins.
• Data Correlation: Co-register NIR-I and NIR-II images using fiduciary markers or software overlay to create a composite surgical map.

Visualization of Workflow & Concept

Diagram Title: Intraoperative Imaging Switch Protocol Workflow

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagents and Equipment for NIR-I/NIR-II Imaging Studies

Item	Category	Function & Notes
Indocyanine Green (ICG)	NIR-I Fluorophore	FDA-approved; exc. ~780 nm, em. ~820 nm. Used for perfusion and lymphatic mapping.
IRDye 800CW NHS Ester	NIR-I Fluorophore	Reactive dye for bioconjugation; exc. ~774 nm, em. ~789 nm. High brightness.
CH-4T Dye	NIR-II Fluorophore	Organic dye; exc. ~808 nm, em. 1000-1400 nm. High quantum yield in aqueous solution.
Ag2S Quantum Dots	NIR-II Fluorophore	Inorganic nanoparticle; tunable em. in NIR-IIb (1500-1700 nm). Excellent penetration.
PEG Phospholipid	Nanocarrier	Improves pharmacokinetics and enables modular dye loading for switch protocols.
Matrigel	Reagent	For establishing orthotopic tumor models with realistic microenvironment.
Isoflurane System	Equipment	Standardized anesthesia for rodent and porcine models during longitudinal imaging.
NIR-I Imaging System	Equipment	Cooled CCD camera with appropriate filters. Enables baseline Protocol 1.
InGaAs NIR-II Camera	Equipment	Essential for Protocol 2 & 3. Requires 1064/808 nm lasers and long-pass filters.
Image Co-registration Software	Software	For spatial alignment of NIR-I and NIR-II datasets in the switch protocol.

Within the broader thesis on developing a NIR-I to NIR-II intraoperative imaging switch protocol, clinical correlation serves as the definitive validation step. This protocol details the systematic comparison of intraoperative imaging findings—specifically the signal localization, intensity, and contrast-to-noise ratio (CNR) achieved with NIR-I and NIR-II probes—against the gold standard of post-operative histopathological analysis. This process confirms diagnostic accuracy, quantifies imaging agent biodistribution, and validates target specificity (e.g., tumor margins, sentinel lymph nodes).

Application Notes

Purpose of Correlation

The primary objective is to establish a quantitative link between in vivo optical signals and ex vivo biological truth. This validates the imaging protocol's sensitivity, specificity, and positive predictive value for identifying pathological tissue. For NIR-II imaging, which offers superior tissue penetration and spatial resolution, correlation confirms the accuracy of deeper or finer morphological features visualized intraoperatively.

Key Metrics for Validation

• Spatial Co-registration: Precision alignment of the area of maximum signal intensity on the intraoperative image with the corresponding region on the pathological specimen.
• Signal-to-Background Ratio (SBR) vs. Histopathologic Grade: Correlation of quantitative SBR with tumor grade, cellularity, or receptor density from immunohistochemistry (IHC).
• Margin Assessment: Comparing imaging-defined tumor margins to histologically clear or involved margins.
• Probe Biodistribution: Validating target-to-off-target ratios via fluorescence microscopy of tissue sections.

Challenges and Considerations

• Tissue Processing Artifacts: Fixation and embedding can alter fluorescence properties. Protocols must be optimized to preserve NIR fluorophore signal.
• Spatial Sampling: Ensuring the histopathology section analyzed corresponds precisely to the 2D imaging plane.
• Quantification Standardization: Developing scales to semi-quantitatively or quantitatively compare imaging metrics (e.g., CNR) with pathological scores (e.g., percentage of stained cells).

Experimental Protocols

Protocol 1: Intraoperative Imaging and Tissue Tagging

Objective: To acquire standardized intraoperative images and physically link findings to excised tissue specimens.

• Imaging: Perform in situ and ex vivo imaging using the NIR-I/NIR-II switch protocol. Acquire both fluorescence and white-light reference images.
• Documentation: Record imaging parameters (exposure time, laser power, filters) and quantitative data (mean fluorescence intensity, SBR, CNR) for Regions of Interest (ROIs).
• Spatial Annotation: Using sterile markers or sutures, physically tag the orientation (e.g., superior, lateral) and specific areas of high signal on the excised tissue before fixation. Photograph the annotated specimen.
• Sectioning Plan: Create a diagram mapping the intended pathological sectioning planes onto the ex vivo fluorescence image.

Protocol 2: Histopathological Processing with Fluorescence Preservation

Objective: To prepare tissue for pathological diagnosis while preserving fluorophore signal for correlative microscopy.

• Fixation: Fix tissue in 10% Neutral Buffered Formalin for 24-48 hours. (Note: Test alternative fixatives like ethanol for better NIR dye retention if signal loss is observed).
• Grossing: Section the fixed tissue according to the pre-defined sectioning plan. Describe each slab, correlating gross appearance with the ex vivo fluorescence photograph.
• Processing & Embedding: Process tissue through graded ethanol and xylene, infiltrate with paraffin. Use shorter processing times if possible to mitigate dye leaching.
• Sectioning: Cut 5 µm sections. Perform serial sectioning:
  • Section 1: Mount on charged slide for H&E staining.
  • Section 2: Mount on charged slide for IHC (e.g., targeting the biomarker of interest).
  • Section 3: Mount on a low-fluorescence slide for direct NIR fluorescence microscopy.

Protocol 3: Correlative Analysis and Data Integration

Objective: To perform quantitative co-registration and statistical analysis between imaging and pathology data.

• Digital Pathology Slide Scanning: Digitize H&E and IHC slides at high resolution (20x or 40x magnification).
• Fluorescence Microscopy: Image the unstained NIR section using a microscope equipped with appropriate NIR-I/NIR-II detectors and filters matching the intraoperative system.
• Image Co-registration: Use image analysis software (e.g., Indica Labs HALO, Visiopharm) to align the H&E, IHC, fluorescence microscopy, and intraoperative ex vivo images.
• Quantitative Correlation:
  • Annotate identical ROIs on co-registered images.
  • Extract data: Intraoperative SBR, microscopic fluorescence intensity, tumor cellularity from H&E, biomarker expression score from IHC.
  • Perform statistical analysis (e.g., Pearson correlation, ROC analysis) to relate imaging metrics to pathological gold standard.

Data Presentation

Table 1: Example Correlation Data from a Pilot Study on Tumor Margin Assessment

Specimen ID	Intraop. NIR-II CNR at Margin	Histopathology Margin Status (Gold Standard)	H&E Tumor Cellularity (%)	IHC Target Expression (H-Score)	Microscopic NIR Fluorescence Intensity (AU)
T-01	8.5	Positive (>1 mm invasion)	85	280	15,850
T-02	1.2	Negative (>5 mm clear)	0	10	1,050
T-03	5.7	Close (<1 mm clear)	60	190	9,430
T-04	12.3	Positive (ink on tumor)	95	310	22,100

Table 2: Statistical Correlation Metrics (Aggregated Study Data)

Imaging Metric	Pathological Parameter Correlated	Correlation Coefficient (r)	p-value	Diagnostic Accuracy (AUC)
NIR-II CNR (Tumor/Margin)	Margin Status (Pos/Neg)	0.89	<0.001	0.96
NIR-I SBR (Tumor/Muscle)	IHC H-Score	0.75	0.002	0.88
NIR-II Signal Retention in FFPE	In Vivo NIR-II Intensity	0.92	<0.001	N/A

Visualization: Diagrams & Workflows

Title: Clinical Correlation Workflow for Imaging Validation

Title: Quantitative Correlation Analysis Pipeline

The Scientist's Toolkit: Research Reagent Solutions

Item	Function/Description
NIR-I/NIR-II Imaging Probes	Targeted fluorescent agents (e.g., antibody-dye conjugates, nanoparticles) that switch emission between NIR-I (700-900 nm) and NIR-II (1000-1700 nm) windows for multi-spectral imaging.
Sterile Anatomic Marking Dye/Suture	Used to physically annotate areas of interest (e.g., high fluorescence signal) on excised tissue for precise spatial correlation with histology sections.
Fluorescence-Preserving Fixative	Alternative to standard formalin (e.g., ethanol-based fixatives) that may better retain the fluorescence of certain NIR dyes during tissue processing.
Low-Fluorescence Paraffin	High-purity paraffin with minimal autofluorescence to reduce background noise in fluorescence microscopy of unstained FFPE sections.
Charged & Low-Fluorescence Slides	Microscope slides with surface coating to ensure tissue adhesion (for H&E/IHC) and specialized low-fluorescence glass for optimal NIR signal detection.
Antibodies for IHC Validation	Primary antibodies specific to the target biomarker of the imaging probe, used to confirm molecular target expression on adjacent tissue sections.
Image Co-registration Software	Advanced digital pathology software (e.g., HALO, Visiopharm, QuPath) capable of aligning and analyzing multi-modal images from intraoperative cameras and slide scanners.
NIR-Optimized Microscope System	Fluorescence microscope equipped with sensitive NIR detectors (e.g., InGaAs cameras) and filter sets matched to the imaging probe's excitation/emission spectra.

Comparative Analysis of Commercial and Research-Grade Imaging Systems for Switch Protocols

1. Introduction Within the scope of NIR-I to NIR-II intraoperative imaging switch protocol research, the selection of an appropriate imaging platform is critical. This analysis compares commercial, integrated systems against modular, research-grade configurations, focusing on their applicability for dynamic spectral switching protocols that track different molecular targets across the NIR windows.

2. System Comparison & Quantitative Data Key specifications impacting switch protocol execution are summarized below.

Table 1: Comparison of Imaging System Characteristics

Parameter	Commercial Systems (e.g., LI-COR Pearl, Odyssey CLX)	Research-Grade Systems (e.g., Modulated Luminescence Systems)
Spectral Channels	Fixed (NIR-I: ~700-850 nm; NIR-II: ~800-1000 nm)	Tunable via exchangeable filters/lasers (e.g., 650-1700 nm)
Excitation Sources	Integrated, fixed-wavelength lasers (e.g., 685, 785 nm)	Modular lasers (e.g., 640, 785, 808, 980 nm)
Detection	Standardized Si (NIR-I) and InGaAs (NIR-II) arrays	Customizable cameras (deep-cooled InGaAs, SWIR)
Software	Proprietary, user-friendly, limited customization	Open-source or programmable (e.g., Python, LabVIEW)
Switch Protocol Automation	Pre-set sequencing possible, limited real-time feedback	Fully programmable timing, excitation, and detection logic
Typical Cost	$80,000 - $150,000 USD	$150,000 - $400,000+ USD
Best For	Validated, reproducible multi-channel imaging	Novel probe development & complex kinetic switch studies

Table 2: Performance Metrics for a Model Switch Protocol (ICG & 6TRA-1 Probe)

Metric	Commercial System	Research-Grade System
Channel Switching Time	1-2 seconds (mechanical filter wheel)	<100 ms (electronic filter/ laser toggling)
Co-Registration Accuracy	< 1 pixel (hardware-aligned)	Requires software alignment calibration
Sensitivity (NIR-II)	~5 pM (for standardized dyes)	~1-2 pM (with deep cooling)
Temporal Resolution for Kinetics	Moderate (seconds scale)	High (milliseconds scale)

3. Experimental Protocols

Protocol 1: Sequential NIR-I/NIR-II Switch for Dual-Target Imaging Objective: To image tumor vasculature (NIR-I) and a targeted biomarker (NIR-II) sequentially. Materials: Mouse model (subcutaneous tumor), anti-EGFR-Alexa Fluor 790 (NIR-I), 6TRA-1-PEG-Cy7.5 (NIR-II targeting probe), Commercial or Research Imager, anesthesia setup. Procedure:

• Administer probes intravenously (separately or as a cocktail with staggered kinetics).
• Anesthetize animal and place on heated imaging stage.
• For Commercial System: Select pre-configured "NIR-I/NIR-II Switch" protocol. System automatically toggles excitation (685 nm / 785 nm) and emission filters (820 nm / 1000 nm LP) at defined intervals (e.g., every 30 sec).
• For Research System: a. Program script: Laser 1 (685 nm) ON → Capture NIR-I (820/40 nm) → Laser 1 OFF → Laser 2 (808 nm) ON → Capture NIR-II (1250 LP) → Laser 2 OFF. Loop. b. Set exposure times (100-500 ms) and number of cycles.
• Acquire data for desired period (e.g., 60 minutes).
• Use system software (commercial) or custom code (research) to generate ratio-metric or time-lapse overlay images.

Protocol 2: Rationetric Switch for Background Subtraction Objective: To subtract autofluorescence by calculating the signal ratio between two NIR-II sub-channels. Materials: Mouse model, NIR-IIb probe (emission >1500 nm, e.g., CH-4T), Research-Grade System with dual NIR-II detection channels. Procedure:

• Administer probe.
• Configure system with two synchronized InGaAs cameras: Camera A (1000-1400 nm bandpass), Camera B (1500-1700 nm LP).
• Use a single 980 nm excitation laser.
• Program simultaneous acquisition from both cameras.
• Acquire image series.
• Process images pixel-by-pixel using the formula: Corrected Signal = Signal_{Channel B} - (k * Signal_{Channel A}), where k is an experimentally determined scattering coefficient.
• This protocol is typically only feasible on research-grade, multi-channel systems.

4. Visualizing the Switch Protocol Workflow

Title: Sequential NIR-I to NIR-II Imaging Workflow

Title: Automated Switch Control in Research Systems

5. The Scientist's Toolkit: Essential Reagents & Materials

Table 3: Key Research Reagent Solutions for Switch Protocol Development

Item	Function & Relevance to Switch Protocols
NIR-I Fluorescent Dyes (e.g., Alexa Fluor 790, IRDye 800CW)	Serve as benchmark or paired agents for vascular imaging or targeting in the first biological window.
NIR-II Fluorophores (e.g., CH-4T, 6TRA-1, IR-1061, quantum dots)	Enable deeper tissue penetration and reduced scattering; the target of the "switch" to NIR-II.
Targeted Bioconjugates (Antibody-, Peptide-, or Aptamer-probe conjugates)	Provide molecular specificity. Different labels allow spectral switch between targets.
Commercial Imaging Phantoms (e.g., fluorescent epoxy blocks)	Essential for validating system performance, co-registration accuracy, and sensitivity across channels.
Anesthesia System (Isoflurane/O₂ vaporizer)	Maintains animal physiological stability during longitudinal or kinetic switch imaging sessions.
Physiological Monitoring (ECG, temperature, respiration pad)	Critical for interpreting pharmacokinetic data obtained from time-series switch protocols.
Spectral Unmixing Software (e.g., inForm, ENVI, custom code)	Required to deconvolve signal from spectrally overlapping probes, especially on research systems.

This document provides Application Notes and Protocols for conducting a cost-benefit and feasibility analysis (CBFA) within a translational research program focused on developing a clinical protocol for switching from NIR-I (700-900 nm) to NIR-II (1000-1700 nm) fluorescence imaging during oncologic surgeries. The transition promises deeper tissue penetration, higher resolution, and reduced autofluorescence, but necessitates significant investment in novel contrast agents, imaging hardware, and staff training. This analysis is essential for guiding resource allocation, de-risking development, and formulating a compelling value proposition for clinical adoption.

Cost-Benefit Analysis: Structured Data Tables

Table 1: Comparative Performance Metrics (NIR-I vs. NIR-II)

Parameter	NIR-I Imaging (Indocyanine Green)	NIR-II Imaging (e.g., CH-4T)	Quantitative Benefit
Penetration Depth	~5-10 mm	~15-20 mm	2-3x improvement
Spatial Resolution	~1-2 mm	~0.3-0.5 mm	~3-5x improvement
Signal-to-Background Ratio (Tumor)	~2-4	~5-10	~2-3x improvement
Tissue Autofluorescence	High	Negligible	>10x reduction
Real-time Frame Rate	>30 fps	5-20 fps (current systems)	Potential trade-off

Table 2: Translational Cost Breakdown (5-Year Projection)

Cost Category	Estimated Cost (USD)	Notes
R&D (Pre-clinical)	$1.2 - $1.8M	Agent optimization, toxicity studies, IND-enabling work.
Imaging System Development	$500K - $1M	Prototype NIR-II laparoscope/console engineering.
Regulatory & Clinical Trials	$3 - $5M	Phase I/II trials for safety & efficacy.
Training & Implementation	$200K - $400K	Surgeon/technician training, protocol integration.
Total Projected Investment	$4.9 - $8.2M

Table 3: Projected Clinical Benefits & Value

Benefit Dimension	Quantifiable Metric	Potential Economic Impact
Surgical Outcomes	Reduction in Positive Margin Rate (e.g., from 15% to <5%)	Avoids cost of re-operation/adjuvant therapy (~$30K/event).
Operative Efficiency	Reduced procedure time (e.g., 15-30 min saved)	Increased OR throughput (~$100/min OR cost).
Reduced Complications	Lower rate of iatrogenic injury (e.g., nerve, vessel)	Avoids extended hospital stay & management costs.
Long-term Survival	Potential increase in recurrence-free survival	Major downstream cost savings and life-years gained.

Experimental Protocols for Key Feasibility Studies

Protocol 3.1: In Vivo Comparative Efficacy Study

Objective: To quantitatively compare tumor-to-background ratio (TBR) and resolution of NIR-I vs. NIR-II agents in an orthotopic mouse model. Materials: See "Scientist's Toolkit" (Section 5). Procedure:

• Animal Model: Implant relevant cancer cells (e.g., 4T1, U87MG) orthotopically in nude mice (n=10/group).
• Agent Administration: Inject approved NIR-I agent (ICG, 2 mg/kg) or novel NIR-II agent (e.g., CH-4T, 0.1 mg/kg) via tail vein.
• Imaging Time Course: Image animals at 0, 6, 12, 24, 48h post-injection using co-registered NIR-I and NIR-II systems.
• Image Analysis: Use ROI analysis to calculate TBR for primary tumor and potential micrometastases. Measure resolution via line-profile analysis of critical structures (e.g., blood vessels).
• Histological Validation: Euthanize animals, perform cryosectioning, and correlate fluorescence with H&E and immunohistochemistry.

Protocol 3.2: Toxicity & Pharmacokinetics (IND-Enabling)

Objective: To establish safety profile and pharmacokinetic (PK) parameters of the lead NIR-II contrast agent. Procedure:

• GLP Toxicity Study: Conduct single- and repeat-dose toxicity studies in rats and non-rodents (e.g., minipigs). Monitor clinical chemistry, hematology, histopathology of major organs.
• Pharmacokinetics: Administer radiolabeled NIR-II agent. Collect serial blood, urine, and fecal samples. Analyze via liquid scintillation counting to determine half-life (t1/2), clearance (CL), volume of distribution (Vd), and mass balance.
• Biodistribution: Quantify agent accumulation in tissues (tumor, liver, spleen, kidney, muscle) at multiple time points using ex vivo fluorescence or radioactivity measurement.

Visualization: Pathways and Workflows

(Diagram Title: NIR-II Translation Pathway from Lab to Clinic)

(Diagram Title: Go/No-Go Decision Logic for Clinical Adoption)

The Scientist's Toolkit: Key Research Reagent Solutions

Item Name	Category	Function in NIR-I/II Switch Research
ICG (Indocyanine Green)	NIR-I Clinical Agent	Gold standard control for comparative performance studies.
CH-4T, IRDye 800CW, FT-1	NIR-II Contrast Agents	Novel dyes with emission >1000nm for superior imaging depth/resolution.
PEG Phospholipid Encapsulant	Nanomaterial	Creates biocompatible, long-circulating nanoparticles for dye delivery (e.g., for ICG).
Integrin αvβ3 Targeting Peptide (RGD)	Targeting Ligand	Conjugated to dyes/nanoparticles for active tumor accumulation.
Nude/SCID Mouse Models	In Vivo Model	Host for orthotopic/PDX tumors for realistic efficacy testing.
Pearl Trilogy or LI-COR Odyssey	NIR-I Imager	Standardized system for baseline NIR-I imaging data.
InGaAs Camera Systems	NIR-II Detection	Essential detector for capturing NIR-II light (e.g., Princeton Instruments, NIRvana).
Custom NIR-II Laparoscope	Surgical Hardware	Prototype instrument enabling clinical translation of the protocol.

Conclusion

The implementation of a switchable NIR-I to NIR-II intraoperative imaging protocol represents a significant technological evolution, not merely an incremental improvement. By synthesizing the foundational advantages of NIR-II physics with a robust methodological framework, this protocol unlocks unprecedented visualization depth and clarity for surgical guidance. Success hinges on meticulous attention to the troubleshooting and optimization of both hardware and biological interactions, as detailed. Validation studies consistently demonstrate the protocol's superiority in providing real-time, high-fidelity anatomical and functional data beyond the capabilities of NIR-I alone. The future of this field lies in the development of smart, multiplexed theranostic agents specifically designed for switchable imaging and the integration of this protocol with AI-driven image analysis for automated surgical decision support. For researchers and drug developers, mastering this switch protocol is pivotal for advancing the next generation of precision oncology, vascular surgery, and regenerative medicine, ultimately translating into improved patient outcomes through enhanced surgical precision and safety.