Journal of Biomedical Optics

SignificanceRapid advances in biophotonics techniques require quantitative, model-based computational approaches to obtain functional and structural information from increasingly complex and multi-scaled anatomies. The lack of efficient tools to accurately model tissue structures and subsequently perform quantitative multi-physics modeling greatly impedes the clinical translation of these modalities. AimWhile the mesh-based Monte Carlo (MMC) method expands our capabilities in simulating complex tissues by using tetrahedral meshes, the generation of such domains often requires specialized meshing tools such as Iso2Mesh. Creating a simplified and intuitive interface for tissue anatomical modeling and optical simulations is essential towards making these advanced modeling techniques broadly accessible to the user community. ApproachWe responded to the above challenge by combining the powerful, open-source 3-D modeling software, Blender, with state-of-the-art 3-D mesh generation and MC simulation tools, utilizing the interactive graphical user interface (GUI) in Blender as the front-end to allow users to create complex tissue mesh models, and subsequently launch MMC light simulations. ResultsHere we present a tutorial to our newly developed Python-based Blender add-on - BlenderPhotonics - to interface with Iso2Mesh and MMC, allowing users to create, configure and refine complex simulation domains and run hardware-accelerated 3-D light simulations with only a few clicks. We provide a comprehensive introduction to this new tool and walk readers through 5 examples, ranging from simple shapes to sophisticated realistic tissue models. ConclusionBlenderPhotonics is user-friendly and open-source, leveraging the vastly rich ecosystem of Blender. It wraps advanced modeling capabilities within an easy-to-use and interactive interface. The latest software can be downloaded at http://mcx.space/bp.

Patient-derived cancer organoids (PDCOs) are a valuable model to recapitulate human disease in culture with important implications for drug development. However, current methods for assessing PDCOs are limited. Label-free imaging methods are a promising tool to measure organoid level heterogeneity and rapidly screen drug response in PDCOs. The aim of this study was to assess and predict PDCO response to treatments based on mutational profiles using label-free wide-field optical redox imaging (WF ORI). WF ORI provides organoid-level measurements of treatment response without labels or additional reagents by measuring the autofluorescence intensity of the metabolic co-enzymes NAD(P)H and FAD. The optical redox ratio is defined as the fluorescence intensity of [NAD(P)H / NAD(P)H +FAD] which measures the oxidation-reduction state of PDCOs. We have implemented WF ORI and developed novel leading-edge analysis tools to maximize the sensitivity and reproducibility of treatment response measurements in colorectal PDCOs. Leading-edge analysis improves sensitivity to redox changes in treated PDCOs (G{Delta} = 1.462 vs G{Delta} = 1.233). Additionally, WF ORI resolves FOLFOX treatment effects across all PDCOs better than two-photon ORI, with [~]7X increase in effect size (G{Delta} = 1.462 vs G{Delta} = 0.189). WF ORI distinguishes metabolic differences based on driver mutations in CRC PDCOs identifying KRAS+PIK3CA double mutant PDCOs vs wildtype PDCOs with 80% accuracy and can identify treatment resistant mutations in mixed PDCO cultures (G{Delta} = 1.39). Overall, WF ORI enables rapid, sensitive, and reproducible measurements of treatment response and heterogeneity in colorectal PDCOs that will impact patient management, clinical trials, and preclinical drug development. Statement of SignificanceLabel-free wide-field optical redox imaging of patient-derived cancer organoids enables rapid, sensitive, and reproducible measurements of treatment response and heterogeneity that will impact patient management, clinical trials, and preclinical drug development.

BACKGROUNDA definitive COVID-19 infection typically is diagnosed by laboratory tests, including real-time, reverse-transcriptase Polymerase Chain Reaction (PCR)-based testing. These currently available COVID-19 tests require the patient to provide an extra-corporeal specimen and the results may not be immediate. Consequently, a variety of rapid antigen tests for COVID-19, all with a wide range of accuracy in terms of sensitivity and specificity, has proliferated (1,2). These rapid tests now represent a significantly larger proportion of all testing done for COVID-19, yet suffer from requiring a physical specimen from the nose or mouth and waiting 15 minutes for most. As a solution, we propose a non-invasive, trans-cutaneous, real-time viral detection device, based on the principles of Raman spectroscopy and machine learning. It does not require any extra-corporeal specimens and can be configured for self-administration. It can be easily used by non-experts and does not require medical training. Our approach suggests that our non-invasive, transcutaneous method may be broadly useful not only in COVID-19 diagnosis, but also in other diagnoses. METHODS160 COVID positive (+) patients and 316 COVID negative (-) patients prospectively underwent nasal PCR testing concurrently with testing using our non-invasive, transcutaneous, immediate viral detector. Both the PCR and our experimental viral detector tests were performed side-by-side on outpatients (N=389) as well as inpatients (N= 87) at Holy Name Medical Center in Teaneck, NJ between June 2021 and August, 2022. The spectroscopic data were generated using an 830nm Raman System with SpectraSoft (W2 Innovations)and then, using machine learning, processed to provide an immediate prediction. A unique patient-interface for finger insertion enabled the application of Raman spectroscopy to viral detection in humans. RESULTSThe data analysis algorithm demonstrates that there is an informative Raman spectrum output from the device, and that individual Raman peaks vary between cases and controls. Our proof-of-concept study yields encouraging results, with a specificity for COVID-19 of 0.75, and a sensitivity (including asymptomatic patients) of 0.80. CONCLUSIONSThe combination of Raman spectroscopy, artificial intelligence, and our unique patient-interface admitting only a patient finger achieved test results of 0.75 specificity and 0.80 sensitivity for COVID-19 testing in this first in human proof-of-concept study. More significantly, the predictability improved with increasing data.

Spinal cord ischemia leads to iatrogenic injury in multiple surgical fields, and the ability to immediately identify onset and anatomic origin of ischemia is critical to its management. Current clinical monitoring, however, does not directly measure spinal cord blood flow, resulting in poor sensitivity/specificity, delayed alerts, and delayed intervention. We have developed an epidural device employing diffuse correlation spectroscopy (DCS) to monitor spinal cord ischemia continuously at multiple positions. We investigate the ability of this device to localize spinal cord ischemia in a porcine model and validate DCS versus Laser Doppler Flowmetry (LDF). Specifically, we demonstrate continuous (>0.1Hz) spatially resolved (3 locations) monitoring of spinal cord blood flow in a purely ischemic model with an epidural DCS probe. Changes in blood flow measured by DCS and LDF were highly correlated (r=0.83). Spinal cord blood flow measured by DCS caudal to aortic occlusion decreased 62%, with a sensitivity of 0.87 and specificity of 0.91 for detection of a 25% decrease in flow. This technology may enable early identification and critically important localization of spinal cord ischemia.

Chemophototherapy (CPT) is an emerging cancer treatment that leverages the synergistic effects of photodynamic therapy (PDT) and chemotherapy. This approach utilizes photosensitizers like Porphyrin Phospholipid (PoP) and Doxorubicin (Dox) to enable phototriggered drug release and targeted tumor destruction. In this study, we present the development and validation of a wide-field laparoscopic spatial frequency domain imaging (SFDI) system, designed to improve intraoperative quantitative fluorescence imaging and monitoring of PoP photobleaching, a PDT-driven effect for tumor destruction, and light-activated Dox release, which facilitates targeted chemotherapeutic drug delivery in an ovarian cancer model. Compared to previous flexible endoscopic imaging methods, our laparoscopic SFDI system offers enhanced spatial coverage, enabling accurate wide-field optical property quantification in minimally invasive surgical settings. Using this system, we performed quantitative fluorescence imaging in vivo to obtain absolute concentrations of PoP and Dox fluorescence, correcting for tissue absorption and scattering effects. This capability allows for precise assessment of PoP photobleaching and Dox release kinetics with improved spatial resolution. Fluorescence imaging revealed a significant reduction in PoP concentration in tumor regions post-illumination, demonstrating the PDT-mediated photobleaching effect and successful light-triggered drug release activation for chemo-induced tumor destruction. The ability to differentiate PoP and Dox fluorescence in a laparoscopic system underscores its potential for real-time intraoperative monitoring of CPT efficacy. These findings establish wide-field laparoscopic SFDI as a promising tool for guiding minimally invasive photodynamic therapy and targeted drug delivery in clinical settings.

SignificanceFluorescence lifetime imaging in the short-wave infrared (SWIR) is expected to enable high resolution multiplexed molecular imaging in highly scattering tissue. AimTo characterize the brightness and fluorescence lifetime of commercially available organic SWIR fluorophores and benchmark them against the tail emission of conventional NIR-excited probes. ApproachCharacterization was performed through our established Time-domain Mesoscopic Fluorescence Molecular Tomography (TD-MFMT) system integrated around a TCSPC-SPAD array. Brightness and fluorescence lifetime was measured for NIR and SWIR probes above 1000 nm. Simultaneous probe imaging was then performed to assess their potential for multiplexed studies. ResultsNIR probes outperformed SWIR probes in brightness while the mean fluorescence lifetimes of the SWIR probes were extremely short. The phantom study demonstrated the feasibility of lifetime multiplexing in the SWIR window with both NIR and SWIR probes. ConclusionsLong tail emission of NIR probes outperformed the SWIR probes in brightness beyond 1000 nm. Fluorescence lifetime was readily detectable in the SWIR window, where the SWIR probes showed shorter lifetimes compared to the NIR probes. We demonstrate the feasibility of lifetime multiplexing in the SWIR window, which paves the way for in vivo multiplexed studies of intact tissues at improved resolution.

SignificanceMitochondria are dynamic organelles that play a key role in energy production and maintaining cellular homeostasis. The regulation of mitochondrial dynamics, involving both fission and fusion, is vital for maintaining a healthy population of mitochondria within the cell. Alterations in mitochondrial dynamics have been associated with various disease states, such as metabolic and neurodegenerative diseases and cancer. AimWe describe a protocol for imaging and analyzing NAD(P)H intensity to visualize the movement of mitochondria over time and in 3D to visualize the distribution of the mitochondrial network within the cell. ApproachA multiphoton (MP) laser scanning microscope was used to image NAD(P)H autofluorescence signal of MDA-MB-231 cells at 750 nm excitation. A mitochondrial fluorescent dye, MitoSpy Orange, was used to validate the signal. Laser power, image size, dwell time, interval time, and imaging duration were optimized for timelapse and 3D imaging to minimize photodamage and maximize autofluorescence signal. ResultsThe NAD(P)H signal in 2D was imaged with a frame rate of 0.4 frames per second (FPS) allowing for visualization of mitochondria movement. 3D imaging was performed with a frame rate of 0.5 FPS for a single cell with a thickness of approximately 15 microns that allowed for visualization of the mitochondria network within the cell. ConclusionsThis protocol motivates using label-free imaging techniques to study mitochondrial dynamics in a non- destructive manner, suitable for drug screening and understanding the effects of mitochondrial dynamic alterations in disease.

SignificanceConventional spectral photoacoustic imaging (sPAI) to assess tissue oxygenation (sO2) uses optical wavelengths in the first near infrared window (NIR-I). This limits the maximum imaging depth ([~]1 cm) due to high spectral coloring of biological tissues. AimSecond near infrared or short-wave infrared (NIR-II or SWIR) wavelengths (950-1400 nm) show potential for deep tissue sPAI due to the exponentially reduced tissue scattering and higher maximum exposure threshold (MPE) in this wavelength range. However, to date, a systematic assessment of NIR-II wavelengths for sPAI of tissue sO2 has yet to be performed. ApproachThe NIR-II PA spectra of oxygenated and deoxygenated hemoglobin was first characterized using a phantom. Optimal wavelengths to minimize spectral coloring were identified. The resulting NIR-II PA imaging methods were then validated in vivo by measuring renal sO2 in adult female rats. ResultssPAI of whole blood under a phantom and of circulating renal blood in vivo, demonstrated PA spectra proportional to wavelength-dependent optical absorption. NIR-II wavelengths had a [~]50% decrease in error of spectrally unmixed blood sO2 compared to conventional NIR-I wavelengths. In vivo measurements of renal sO2 validated these findings and demonstrated a [~]30% decrease in error of estimated renal sO2 when using NIR-II wavelengths for spectral unmixing in comparison to NIR-I wavelengths. ConclusionssPAI using NIR-II wavelengths improved the accuracy of tissue sO2 measurements. This is likely due to the overall reduced spectral coloring in this wavelength range. Combined with the increased safe skin exposure fluence limits in this wavelength range, demonstrate the potential to use NIR-II wavelengths for quantitative sPAI of sO2 from deep heterogeneous tissues.

SignificanceCollagen autofluorescence provides valuable intrinsic contrast for assessing tissue structure, composition, and pathology. However, a comprehensive understanding of the fluorescence properties across different collagen types remains limited. This knowledge gap may limit the development of advanced label-free fluorescence spectroscopy and imaging techniques for specific tissue characterization and diagnostic applications. AimThis study aims to comprehensively characterize the fluorescence intensity excitation-emission matrices (I-EEMs) and time-resolved excitation-emission matrices (TR-EEMs) of collagen standards from Types I, II, III, IV, and V obtained from various organ sources under both dry and hydrated conditions, to identify optimal excitation-emission parameters for each collagen type discrimination, and to establish a reference dataset that supports future research in label-free tissue characterization. ApproachWe employed a time-resolved fluorescence spectroscopy system equipped with an optical parametric oscillator laser (excitation: 200-2000 nm, pulse width: 30 ps) as an excitation source to generate I-EEMs and TR-EEMs of human and bovine collagen Types I-V. The fluorescence light was obtained by a multichannel plate photomultiplier tube through a monochromator (spectral range: 200-1000 nm). Measurements were conducted using collagen standards, under both dry and hydrated states. Additionally, photobleaching effects were assessed to ensure the reliability and reproducibility of fluorescence data. ResultsEach collagen type exhibited distinct I-EEM and TR-EEM signatures, with fluorescence lifetimes ranging from 2.5 ns (Type III, bovine skin) to 5.3 ns (Types II and V). Fibrillar collagens (Types I and V) displayed broader I-EEMs, whereas basement membrane collagen (Type IV) showed the narrowest spectral distribution. Organ-source-dependent variations were evident within the same collagen type. Type I collagen from human placenta exhibited an inverse lifetime-emission wavelength relationship compared to bovine sources. Hydration consistently red-shifted emission peaks into the 395-420 nm range and reduced fluorescence lifetimes across all collagen types (e.g., Type I bovine Achilles tendon: 3.2-5.0 ns dry vs. 3.0-4.5 ns hydrated). Despite excitation wavelength- and fluence-dependent photobleaching of fluorescence intensity, fluorescence lifetimes remained relatively stable, confirming the robustness of lifetime-based measurements. ConclusionsThis study establishes a comprehensive reference dataset for the fluorescence properties of collagen Types I-V and demonstrates the potential of combined I-EEMs and TR-EEMs analysis for tissue characterization. The results highlight species-, organ-, type-, and environment-specific optical fingerprints of similar collagens, which must be considered before implementing more in-depth studies on how the optical properties of collagen change in different medical applications.

We present a light-emitting diode (LED)-based transcranial photoacoustic measurement (LED-trPA) of oxyhemoglobin (HbO2) saturation at superior sagittal sinus (SSS) in hypoxic neonatal piglets. The optimal LED imaging wavelengths and frame averaging scheme were determined based on in vivo characterization of transcranial sensitivity. Based on the framework (690/850 nm with >20 frame averaging), graded hypoxia was successfully identified in neonatal piglets in vivo with less than 10.0 % of root mean squared error (RMSE). This preclinical study suggests the feasibility of a rapid, cost-effective, and safe LED-trPA monitoring of perinatal hypoxia-ischemia and prompt interventions for clinical use.

Melanin plays a significant role in the regulation of epidermal homeostasis and photoprotection of human skin. The assessment of its epidermal distribution and overall content is of great interest due to its involvement in a wide range of physiological and pathological skin processes. Among several spectroscopic and optical imaging methods that have been reported for non-invasive quantification of melanin in human skin, the approach based on the detection of two-photon excited fluorescence lifetime distinguishes itself by enabling selective detection of melanin with sub-cellular resolution, thus facilitating its quantification while also resolving its depth-profile. A key limitation of prior studies on the melanin assessment based on this approach is their inability to account for the skin heterogeneity due to the reduced field of view of the images, which results in high dispersion of the measurement values. Pigmentation in both normal and pathological human skin is highly heterogeneous and its macroscopic quantification is critical for reliable measurements of the epidermal melanin distribution and for capturing melanin-related sensitive dynamic changes as a response to treatment. In this work, we employ a fast large-area multiphoton exoscope (FLAME), recently developed by our group for clinical skin imaging, that has the ability to evaluate the 3D distribution of epidermal melanin content in vivo macroscopically (millimeter scale) with microscopic resolution (sub-micron) and rapid acquisition rates (minutes). We demonstrate significant enhancement in the reliability of the melanin density and distribution measurements across Fitzpatrick skin types I to V by capturing the intra-subject pigmentation heterogeneity enabled by the large volumetric sampling. We also demonstrate the potential of this approach to provide consistent measurement results when imaging the same skin area at different times. These advances are critical for clinical and research applications related to monitoring pigment modulation as a response to therapies against pigmentary skin disorders, skin aging, as well as skin cancers.

SignificanceHigh-quality photoplethysmography (PPG) signals are essential for accurate extraction of cardiovascular metrics such as heart rate, heart rate variability, and perfusion index. However, signal degradation for individuals with dark skin tones can compromise PPG quality and pose challenges for equitable sensing. AimWe develop a dual-wavelength, polarization-sensitive PPG device to assess perfusion index (PI) across a range of skin tones. ApproachTo evaluate the impact of polarization on PPG signal quality, we record PI for co-polarized (polarized illumination and parallel-aligned polarized detection), and cross-polarized conditions (polarized illumination and orthogonally aligned polarized detection) at 655 nm and 940 nm in participants representing light, medium, and brown skin tone categories. Skin tone classification are based on the individual typology angle (ITA) values derived from the CIE L*b* color space measurements. ResultsAt 940 nm, light from the cross-polarized light channel significantly increases PI (p < 0.05). At 655 nm, cross-polarization presents a statistically significantly enhanced PI (p < 0.05) relative to light from the co-polarized illumination condition, although the magnitude of the improvement decreases with lighter skin tone indication a possible interaction between skin tone and polarization. This improvement is consistent across all skin tones. ConclusionsOur results suggest that the cross-polarized condition improves PPG signal quality by reducing the influence of superficial scattering and enhancing deeper vascular signals. This approach may be especially beneficial for individuals with darker skin tones and offers a promising path towards more robust and inclusive physiological monitoring using PPG-based technologies.

Background and objectivesVisible-light optical coherence tomography (vis-OCT) has enabled the visualization of retinal structures and functions beyond the capabilities of conventional OCTs. However, to reconstruct high-quality images, vis-OCT requires special post-processing, including balanced detection. An open-source, standardized vis-OCT data processing software is essential for clinical applications and translation of vis-OCT. MethodsWe developed Vis-OCT Explorer, an open-source, modular Python-based software for processing vis-OCT images. In addition to the standard spectral-domain OCT processing pipeline - including k-space resampling, dispersion compensation, and fast Fourier transformation - Vis-OCT Explorer offers unique dual-spectrometer balanced detection, short-time-Fourier transformation (STFT) based dispersion compensation coefficient optimization, and GPU-accelerated processing. We evaluated the reconstruction performance by quantifying a quality index extracted from individual B-scan images. We also assessed the repeatability of retinal thickness measurements by five operators on images acquired from different testing sites using the intraclass correlation coefficient (ICC) analysis. ResultsBalanced detection and STFT-based dispersion compensation significantly increased the quality index of reconstructed B-scan images. ICC values of the retinal nerve fiber layer (RNFL) and ganglion cell-inner plexiform layer (GCIPL) thickness measurements from four testing sites exceeded 0.8 in 87.5% of the macular-centered images. The ICC of RNFL thickness measurements on all optic nerve head-centered images is above 0.8, showing strong repeatability across users. ConclusionsVis-OCT Explorer provides high-quality image processing and enables highly repeatable measurements on vis-OCT human retinal images. It facilitates future multicenter clinical tests to validate vis-OCTs clinical efficacy.

Recently, skin pigmentation has been shown to affect the performance of pulse oximeters and other light-based techniques like photoacoustic imaging, tissue oximetry, and continuous wave near infrared spectroscopy. Evaluating the robustness to changes in skin pigmentation is therefore essential for the proper use of optical technologies in the clinical scenario. We conducted systematic time domain near infrared spectroscopy measurements on calibrated tissue phantoms and in vivo on volunteers during static and dynamic (i.e., arterial occlusion) measurements. To simulate varying melanosome volume fractions in the skin, we inserted, between the target sample and the measurement probe, thin tissue phantoms made of silicone and nigrosine (skin phantoms). Additionally, we conducted an extensive measurement campaign on a large cohort of pediatric subjects, covering the full spectrum of skin pigmentation. Our findings consistently demonstrate that skin pigmentation has a negligible effect on time domain near infrared spectroscopy results, underscoring the reliability and potential of this emerging technology in diverse clinical settings.

SignificanceWhile shortwave infrared (SWIR) imaging provides superior tissue penetration and reduced autofluorescence for preclinical applications, quantitative fluorescence analysis is hindered by the limited dynamic range of InGaAs cameras, forcing a focus on either bright or dim anatomical features. AimWe develop a high dynamic range (HDR) imaging method specifically adapted for the high-noise characteristics of InGaAs detectors to enable quantitative fluorescence imaging across wide intensity ranges. We demonstrate that one-time camera calibration based on a series of images encompassing the range of radiance intensities enables all subsequent image processing. ApproachWe modified classical HDR algorithms with exposure-time-dependent dark current subtraction, preprocessing to exclude saturated and noisy pixels before camera response function recovery, and dynamic weighting range adjustment to account for shrinking intensity ranges at longer exposures. HDR image processing effects on preclinical imaging outcomes were analyzed using indocyanine green and SWIR-emitting PbS/CdS quantum dots in mouse models. ResultsHDR imaging achieved a 22 dB improvement in dynamic range over single exposures, enabling simultaneous quantification across more than three orders of magnitude of fluorophore concentration. In vivo studies showed improvements in contrast-to-noise ratios across all anatomical features, with improvements in vascular contrast while maintaining quantitative accuracy. After one-time camera calibrations, this approach enables rapid processing of subsequent datasets. ConclusionsThis software-based HDR SWIR imaging approach eliminates exposure parameter optimization and enables comprehensive biodistribution analysis across all anatomical structures from a single acquisition sequence, significantly streamlining preclinical imaging workflows while preserving quantitative accuracy.

This study proposes a novel optical method of detecting and reducing SARS-CoV-2 transmission, the virus responsible for the COVID-19 pandemic that is sweeping the world today. SARS-CoV-2 belongs to the {beta}-coronaviruses characterized by the crown-shaped spike protein that protrudes out of the virus particles, giving the virus a "corona" shape; hence the name coronavirus. This virus is similar to the viruses that caused SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome), the other two coronavirus epidemics that were recently contained within the last ten years. The technique being proposed uses a light source from a smart phone and a mobile spectrophotometer to enable detection of viral proteins in solution or paper as well as protein-protein interactions. The proof-of-concept is shown by detecting soluble preparations of spike protein subunits from SARS-CoV-2, followed by detection of the actual binding potential of the spike protein with its host receptor, the angiotensin-converting enzyme 2 (ACE2). The results are validated by showing that this method can detect antigen-antibody binding using two independent viral protein-antibody pairs. The binding could be detected optically both in solution and on a solid support such as nitrocellulose membrane. Finally, this technique is combined with DC bias to show that introduction of a current into the system can be used to disrupt the antigen-antibody reaction, suggesting that the proposed extended technique can be a potential means of not only detecting the virus, but also reducing virus transmission by disrupting virus-receptor interactions electrically. SignificanceThe measured intensity of light can reveal information about different cellular parameters under study. When light passes through a bio-composition, the intensity is associated with its content. The nuclei size, cell shape and the refractive index variation of cells contributes to light intensity. In this work, an optical label-free real time detection method incorporating the smartphone light source and a portable mini spectrometer for SARS-CoV-2 detection was developed based on the ability of its spike protein to interact with the ACE2 receptor. The light interactions with control and viral protein solutions were capable of providing a quick decision regarding whether the sample under test was positive or negative, thus enabling SARS-CoV-2 detection in a rapid manner.

Microcirculatory dysfunction in dermal (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans may predict cardio-metabolic disease progression. In-vivo visualization and monitoring of microvascular remodeling in these tissues remains challenging. We compared performance of multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT of diet-induced obese (DIO) mice undergoing voluntary wheel running. MSOT quantitatively visualized lipid and hemoglobin contrast in fat depots at early stages of DIO. RSOM precisely visualizes microvasculature with quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. Combination of MSOT and RSOM precisely monitor microcirculatory dysfunction and intervention response in dWAT and scWAT of DIO mice. Our findings lay out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in cardio-metabolic disease.

SignificanceChanges in interstitial fluid clearance are implicated in many diseases. Using NIR imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies. AimWe investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces. ApproachWe used a chemically inert IR Dye 800CW (free dye) to target venous capillaries and a purified conjugate of IR Dye 680RD with a 40 kDa PEG (PEG) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imagings utility in two in vivo tissue beds - the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise. ResultsIn an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. Co-injected NIR tracers cleared from the interstitial space via distinct routes allowed assessment lymphatic and venous uptake in the mouse tail. We determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, while exercise pre-injection exhibited a transient delay in clearance from the joint ConclusionsNIR imaging enables of simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure transient changes in clearance rates and pathways.

Our research is focused on creating and simulating hyper-realistic artificial human tissue analogues. Generation and simulation of macroscopic biological material depends upon accurate ground-truth data on spectral properties of materials. Here, we developed methods for high fidelity spectral data collection using two differently colored simulated skin tissue samples and a portable spectral imaging camera. Using the standard procedure, we developed, we quantified the reproducibility of the spectral image signatures of the two synthetic skin samples under natural and artificial lighting conditions commonly found in clinical settings. We found high coefficients of determination for all measures taken under the same lighting. As expected, we found the spectral image signature of each sample was dependent on the illumination source. Our results confirm that illumination spectra data should be included with spectral image data. The high-fidelity methods for spectral image data collection we developed here should facilitate accurate collection of spectral image signature data for gross biological samples and synthetic materials collected under the same illumination source.

Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTS is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy (MPM) is capable of capturing high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence) through two-photon excited fluorescence (2PEF). Second harmonic generation (SHG) is another popular method of generating image contrast with MPM and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to autofluorescence change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, angiogenesis). MPM was used to image 12 separate patient samples of formalin-fixed and paraffinized DGAST slides with a SHG channel 4 2PEF channels, each tuned to capture fluorescence from NADH, FAD, lipofuscin, and porphyrin. We found that there was a significant difference in the relative abundance of signal generated in the 2PEF in comparison to the neighboring tissues of the duodenum. Texture extraction was used to create linear discriminant classifiers for tumor vs all other tissue classes before and after principal component analysis (PCA) of the texture feature dataset. PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy of the classifier. The LDA classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6 - 93.8%. These results suggest that MPM 2PEF and SHG imaging is a promising label-free method for discriminating between DGAST tumors and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.