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Significance: Although the lymphatic system is the second largest circulatory system in the body, there are limited techniques available for characterizing lymphatic vessel function. We report shortwave-infrared (SWIR) imaging for minimally invasive in vivo quantification of lymphatic circulation with superior contrast and resolution compared with near-infrared first window imaging. Aim: We aim to study the lymphatic structure and function in vivo via SWIR fluorescence imaging. Approach: We evaluated subsurface lymphatic circulation in healthy, adult immunocompromised salt-sensitive Sprague--Dawley rats using two fluorescence imaging modalities: near-infrared first window (NIR-I, 700 to 900 nm) and SWIR (900 to 1800 nm) imaging. We also compared two fluorescent imaging probes: indocyanine green (ICG) and silver sulfide quantum dots (QDs) as SWIR lymphatic contrast agents following intradermal footpad delivery in these rats. Results: SWIR imaging exhibits reduced scattering and autofluorescence background relative to NIR-I imaging. SWIR imaging with ICG provides 1.7 times better resolution and sensitivity than NIR-I, and SWIR imaging with QDs provides nearly two times better resolution and sensitivity with enhanced vessel distinguishability. SWIR images thus provide a more accurate estimation of in vivo vessel size than conventional NIR-I images. Conclusions: SWIR imaging of silver sulfide QDs into the intradermal footpad injection provides superior image resolution compared with conventional imaging techniques using NIR-I imaging with ICG dye. [ABSTRACT FROM AUTHOR]

Significance: Necrotizing soft-tissue infections (NSTIs) are life-threatening infections with a cumulative case fatality rate of 21%. The initial presentation of an NSTI is non-specific, frequently leading to misdiagnosis and delays in care. No current strategies yield an accurate, real-time diagnosis of an NSTI. Aim: A first-in-kind, observational, clinical pilot study tested the hypothesis that measurable fluorescence signal voids occur in NSTI-affected tissues following intravenous administration and imaging of perfusion-based indocyanine green (ICG) fluorescence. This hypothesis is based on the established knowledge that NSTI is associated with local microvascular thrombosis. Approach: Adult patients presenting to the Emergency Department of a tertiary care medical center at high risk for NSTI were prospectively enrolled and imaged with a commercial fluorescence imager. Single-frame fluorescence snapshot and first-pass perfusion kinetic parameters--ingress slope (IS), time-to-peak (TTP) intensity, and maximum fluorescence intensity (IMAX)--were quantified using a dynamic contrast-enhanced fluorescence imaging technique. Clinical variables (comorbidities, blood laboratory values), fluorescence parameters, and fluorescence signal-to-background ratios (SBRs) were compared to final infection diagnosis. Results: Fourteen patients were enrolled and imaged (six NSTI, six cellulitis, one diabetes mellitus-associated gangrene, and one osteomyelitis). Clinical variables demonstrated no statistically significant differences between NSTI and non-NSTI patient groups (p-value ≥ 0.22). All NSTI cases exhibited prominent fluorescence signal voids in affected tissues, including tissue features not visible to the naked eye. All cellulitis cases exhibited a hyperemic response with increased fluorescence and no distinct signal voids. Median lesion-to-background tissue SBRs based on snapshot, IS, TTP, and IMAX parameter maps ranged from 3.2 to 9.1, 2.2 to 33.8, 1.0 to 7.5, and 1.5 to 12.7, respectively, for the NSTI patient group. All fluorescence parameters except TTP demonstrated statistically significant differences between NSTI and cellulitis patient groups (p-value < 0.05). Conclusions: Real-time, accurate discrimination of NSTIs compared with nonnecrotizing infections may be possible with perfusion-based ICG fluorescence imaging. [ABSTRACT FROM AUTHOR]

Significance: Head and neck squamous cell carcinoma (HNSCC) has a particularly poor prognosis. Improving the surgical resection boundary, reducing local recurrence, and ultimately ameliorating the overall survival rate are the treatment goals. Aim: To obtain a complete surgical resection (R0 resection), we investigated the use of a fluorescent imaging probe that targets the integrin subtype αvβ6, which is upregulated in many kinds of epithelial cancer, using animal models. Approach: αvβ6 expression was detected using polymerase chain reaction (PCR) and immunoprotein blotting of human tissues for malignancy. Protein expression localization was observed. αvβ6 and epidermal growth factor receptor (EGFR) were quantified by PCR and immunoprotein blotting, and the biosafety of targeting the αvβ6 probe material was examined using Cell Counting Kit-8 assays. Indocyanine green (ICG) was used as a control to determine the localization of the probe at the cellular level. In vivo animal experiments were conducted through tail vein injections to evaluate the probe’s imaging effect and to confirm its targeting in tissue sections. Results: αvβ6 expression was higher than EGFR expression in HNSCC, and the probe showed good targeting in in vivo and in vitro experiments with a good safety profile. Conclusions: The ICG-αvβ6 peptide probe is an exceptional and sensitive imaging tool for HNSCC that can distinguish among tumor, normal, and inflammatory tissues. [ABSTRACT FROM AUTHOR]

Significance: Near-infrared fluorescence imaging still lacks a standardized, objective method to evaluate fluorescent dye efficacy in oncological surgical applications. This results in difficulties in translation between preclinical to clinical studies with fluorescent dyes and in the reproduction of results between studies, which in turn hampers further clinical translation of novel fluorescent dyes. Aim: Our aim is to develop and evaluate a semi-automatic standardized method to objectively assess fluorescent signals in resected tissue. Approach: A standardized imaging procedure was designed and quantitative analysis methods were developed to evaluate non-targeted and tumor-targeted fluorescent dyes. The developed analysis methods included manual selection of region of interest (ROI) on white light images, automated fluorescence signal ROI selection, and automatic quantitative image analysis. The proposed analysis method was then compared with a conventional analysis method, where fluorescence signal ROIs were manually selected on fluorescence images. Dice similarity coefficients and intraclass correlation coefficients were calculated to determine the inter- and intraobserver variabilities of the ROI selections and the determined signal- and tumor-tobackground ratios. Results: The proposed non-targeted fluorescent dyes analysis method showed statistically significantly improved variabilities after application on indocyanine green specimens. For specimens with the targeted dye SGM-101, the variability of the background ROI selection was statistically significantly improved by implementing the proposed method. Conclusion: Semi-automatic methods for standardized quantitative analysis of fluorescence images were successfully developed and showed promising results to further improve the reproducibility and standardization of clinical studies evaluating fluorescent dyes. [ABSTRACT FROM AUTHOR]

Significance: Indocyanine green-based dynamic contrast-enhanced fluorescence imaging (DCE-FI) can objectively assess bone perfusion intraoperatively. However, it is susceptible to motion artifact due to patients' involuntary respiration and mechanical disturbance. Reducing motion artifacts would significantly improve DCE-FI for orthopedic surgical guidance. Aim: Our primary objective is to develop an automated correction method to reduce motion artifacts in DCE-FI and improve the accuracy of bone perfusion assessment. Approach: We developed an automated motion correction approach based on frame-by-frame mutual information (MI) and validated the effectiveness of this approach in various phantom studies and patient images from 45 imaging sessions of fifteen amputees. Results: The MI-based correction reduced motion artifacts by 93% for mechanical disturbances and 76% for simulated respiration in phantom studies. Patient images show improved alignment, improved kinetic curves, and restored bone perfusionrelated parameters with an average correction of 4.3 and 9.6 mm in x- and y-axes per session. Conclusions: The automated MI-based motion correction was able to eliminate motion artifacts effectively and significantly improved the quantitative assessment of bone perfusion by DCE-FI. [ABSTRACT FROM AUTHOR]

Fluorescence guided surgery (FGS) has demonstrated improvements in decision making and patient outcomes for a wide range of surgical procedures. Not only can FGS systems provide a higher level of structural perfusion accuracy in tissue reconstruction cases but they can also serve for real-time functional characterization. Multiple FGS devices have been Food and Drug administration (FDA) cleared for use in open and laparoscopic surgery. Despite the rapid growth of the field, there has been a lack standardization methods. This work overviews commonalities inherent to optical imaging methods that can be exploited to produce such a standardization procedure. Furthermore, a system evaluation pipeline is proposed and executed through the use of photo-stable indocyanine green fluorescence phantoms. Five different FDA-approved open-field FGS systems are used and evaluated with the proposed method. The proposed pipeline encompasses the following characterization: (1) imaging spatial resolution and sharpness, (2) sensitivity and linearity, (3) imaging depth into tissue, (4) imaging system DOF, (5) uniformity of illumination, (6) spatial distortion, (7) signal to background ratio, (8) excitation bands, and (9) illumination wavelength and power. The results highlight how such a standardization approach can be successfully implemented for inter-system comparisons as well as how to better understand essential features within each FGS setup. Despite clinical use being the end goal, a robust yet simple standardization pipeline before clinical trials, such as the one presented herein, should benefit regulatory agencies, manufacturers, and end-users to better assess basic performance and improvements to be made in next generation FGS systems. [ABSTRACT FROM AUTHOR]

Preclinical fluorescence imaging with NIR-I (700 to 900 nm) illumination and short-wave infrared or NIR-II (1000 to 1700 nm) emission increases tissue penetration depth and improves resolution through decreased scattering. Background autofluorescence decreases signal-to-background ratios (SBR) in fluorescence imaging; maximizing SBR will further improve the impact of deep tissue imaging. The impact of rodent diet, illumination wavelength, and emission range on the background fluorescence and contrast agent SBR were determined to assist with the experimental design of future imaging studies. Following illumination with 670, 760, or 808 nm, autofluorescence in the NIR-I (<975 nm), NIR-II (>1000 nm), and NIR-II LP (>1250 nm) regions was assessed in mice fed chow or a purified diet using an IR VIVO preclinical imager (Photon, Etc.). Comparison of the SBR of liver-localized indocyanine green in the various imaging conditions indicated when gut autofluorescence was a problematic confounder. Mice fed chow exhibit high levels of background autofluorescence in the gastrointestinal tract and, to a lesser extent, skin when illuminated with 670 nm light for NIR-I imaging (700 to 975 nm), interfering with the identification of fluorescently labeled tissue. Background autofluorescence was reduced by more than two orders of magnitude by any of the following changes: (1) purified diet; (2) excitation with 760 or 808 nm illumination; or (3) emission in the NIR-II (1000 to 1600 or 1250 to 1600 nm). Although the SBR was generally sufficient for feature identification except when imaging of chow-fed mice with 670 nm excitation and NIR-I emission, switching to a purified diet, using longer excitation wavelengths, or using longer emission wavelengths improved SBR significantly. Systematic comparison of imaging conditions and diet highlights the reduction in autofluorescence and increase in SBR enabled by intentional choices in the experimental parameters including diet, excitation wavelength, and emission wavelength range. [ABSTRACT FROM AUTHOR]

Pancreatic cancer tumors are known to be avascular, but their neovascular capillaries are still chaotic leaky vessels. Capillary permeability could have significant value for therapy assessment, and its quantification might be possible with macroscopic imaging of indocyanine green (ICG) kinetics in tissue. The capacity of using standard fluorescence surgical systems for ICG kinetic imaging as a probe for capillary leakage was evaluated using a clinical surgical fluorescence imaging system, as interpreted through vascular permeability modeling. Xenograft pancreatic adenocarcinoma models were imaged in mice during bolus injection of ICG to capture the kinetics of uptake. Image analysis included ratiometric data, normalization, and match to theoretical modeling. Kinetic data were converted into the extraction fraction of the capillary leakage. Pancreatic tumors were usually less fluorescent than the surrounding healthy tissues, but still the rate of tumor perfusion could be assessed to quantify capillary extraction. Model simulations showed that flow kinetics stabilized after about 1 min beyond the initial bolus injection and that the relative extraction fraction model estimates matched the experimental data of normalized uptake within the tissue. The kinetics in the time period of 1 to 2 min post-injection provided optimal differential data between AsPC1 and BxPC3 tumors, although high individual variation exists between tumors. ICG kinetic imaging during the initial leakage phase was diagnostic for quantitative vascular permeability within pancreatic tumors. Methods for autogain correction and normalized model-based interpretation allowed for quantification of extraction fraction and difference identification between tumor types in early timepoints. [ABSTRACT FROM AUTHOR]

As clinical evidence on the colorectal application of indocyanine green (ICG) perfusion angiography accrues, there is also interest in computerizing decision support. However, user interpretation and software development may be impacted by system factors affecting the displayed near-infrared (NIR) signal. We aim to assess the impact of camera positioning on the displayed NIR signal across different open and laparoscopic camera systems. The effects of distance, movement, and target location (center versus periphery) on the displayed fluorescence signal of different systems were measured under electromagnetic stereotactic guidance from an ICG-albumin model and in vivo during surgery. Systems displayed distinct fluorescence performances with variance apparent with scope optical lens configuration (0 deg versus 30 deg), movement, target positioning, and distance. Laparoscopic system readings fitted inverse square function distance-intensity curves with one device and demonstrated a direction dependent sigmoid curve. Laparoscopic cameras presented central targets as brighter than peripheral ones, and laparoscopes with angled optical lens configurations had a diminished field of view. One handheld open system also showed a distance-intensity relationship, whereas the other maintained a consistent signal despite distance, but both presented peripheral targets brighter than central ones. Optimal clinical use and signal computational development requires detailed appreciation of system behaviors. [ABSTRACT FROM AUTHOR]

Significance: Fluorescence imaging of rheumatoid diseases with indocyanine green (ICG) is an emerging technique with unique potential for diagnosis and therapy. Device characterization, monitoring of the performance, and further developments of the technique require tissue-equivalent fluorescent phantoms of high stability with appropriate anatomical shapes. Aim: Our investigations aim at the development of a three-dimensional (3D) printing technique to fabricate hand and finger models with appropriate optical properties in the near-infrared spectral range. These phantoms should have fluorescence properties similar to ICG, and excellent photostability and durability over years. Approach: We modified a 3D printing methacrylate photopolymer by adding the fluorescent dye Lumogen IR 765 to the raw material. Reduced scattering and absorption coefficients were adjusted to values representative of the human hand by incorporating titanium dioxide powder and black ink. The properties of printed phantoms of various compositions were characterized using UV/Vis and fluorescence spectroscopy, and time-resolved measurements. Photostability and bleaching were investigated with a hand imager. For comparison, several phantoms with ICG as fluorescent dye were printed and characterized as well. Results: The spectral properties of Lumogen IR 765 are very similar to those of ICG. By optimizing the concentrations of Lumogen, titanium dioxide, and ink, anatomically shaped hand and vessel models with properties equivalent to in vivo investigations with a fluorescence hand imager could be printed. Phantoms with Lumogen IR 765 had an excellent photostability over up to 4 years. In contrast, phantoms printed with ICG showed significant bleaching and degradation of fluorescence over time. Conclusions: 3D printing of phantoms with Lumogen IR 765 is a promising method for fabricating anatomically shaped fluorescent tissue models of excellent stability with spectral properties similar to ICG. The phantoms are well-suited to monitor the performance of hand imagers. Concepts can easily be transferred to other fluorescence imaging applications of ICG. [ABSTRACT FROM AUTHOR]

Significance: X-ray imaging serves as the mainstream imaging in dentistry, but it involves risk of ionizing radiation. Aim: This study presents the feasibility of indocyanine green-assisted near-infrared fluorescence (ICG-NIRF) dental imaging with 785-nm NIR laser in the first (ICG-NIRF-I: 700 to 1000 nm) and second (ICG-NIRF-II: 1000 to 1700 nm) NIR wavelengths. Approach: Sprague Dawley rats with different postnatal days were used as animal models. ICG, as a fluorescence agent, was delivered to dental structures by subcutaneous injection (SC) and oral administration (OA). Results: For SC method, erupted and unerupted molars could be observed from ICG-NIRF images at a short imaging time (<1 min). ICG-NIRF-II could achieve a better image contrast in unerupted molars at 24 h after ICG injection. The OA could serve as a non-invasive method for ICG delivery; it could also cause the glow-in-dark effect in unerupted molars. For erupted molars, OA can be considered as mouthwash and exhibits outstanding performance for delivery of ICG dye; erupted molar structures could be observed at a short imaging time (<1 min) and low ICG dose (0.05 mg / kg). Conclusions: Overall, ICG-NIRF with mouthwash could perform in-vivo dental imaging in two NIR wavelengths at a short time and low ICG dose. [ABSTRACT FROM AUTHOR]

Significance: Dynamic diffuse fluorescence tomography (DFT) can recover the static distribution of fluorophores and track dynamic temporal events related to physiological and disease progression. Dynamic imaging indocyanine green (ICG) approved by the food and drug administration is still under-exploited because of its characteristics of low quantum yield and relatively rapid tissue metabolism. Aim: In order to acquire the ICG tomographic image sequences for pharmacokinetic analysis, a dynamic DFT system was proposed. Approach: A fiber-based dynamic DFT system adopts square-wave modulation lock-in photon-counting scheme and series-parallel measurement mode, which possesses high sensitivity, large dynamic range, high anti-ambient light ability in common knowledge, as well as good cost performance. In order to investigate the effectiveness of the proposed system, the measurement stability and the anti-crosstalk—a crucial factor affecting the system parallelization—were assessed firstly, then a series of static phantoms, dynamic phantoms and in vivo mice experiments were conducted to verify the imaging capability. Results: The system has the limited dynamic range of 100 dB, the fluctuation of photon counting within 3%, and channel-to-channel crosstalk ratio better than 1.35. Under the condition of a sufficient signal-to-noise ratio, a complete measurement time for one frame image was 10.08 s. The experimental results of static phantoms with a single target and three targets showed that this system can accurately obtain the positions, sizes, and shapes of the targets and the reconstructed images exhibited a high quantitativeness. Further, the self-designed dynamic phantom experiments demonstrated the capability of the system to capture fast changing fluorescence signals. Finally, the in vivo experiments validated the practical capability of the system to effectively track the ICG metabolism in living mice. Conclusions: These results demonstrate that our proposed system can be utilized for assessing ICG pharmacokinetics, which may provide a valuable tool for tumor detection, drug assessment, and liver function evaluation. [ABSTRACT FROM AUTHOR]