Live-Cell Imaging Quantifies Changes in Function and Metabolic NADH Autofluorescence During Macrophage-Mediated Phagocytosis of Tumor Cells.

BACKGROUND: The immune system has evolved to detect foreign antigens and deliver coordinated responses, while minimizing "friendly fire." Until recently, studies investigating the behavior of immune cells were limited to static in vitro measurements. Although static measurements allow for real-time imaging, results are often difficult to translate to an in vivo setting. Multiphoton microscopy is an emerging method to capture spatial information on subcellular events and characterize the local microenvironment. Previous studies have shown that multiphoton microscopy can monitor changes in single-cell macrophage heterogeneity during differentiation. Therefore, there is a need to use multiphoton microscopy to monitor molecular interactions during immunological activities like phagocytosis. Here we investigate the correlation between phagocytic function and changes in endogenous optical reporters during phagocytosis. METHODS: In vitro autofluorescence imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) was used to detect metabolic changes in macrophages during phagocytosis. More specifically, optical redox ratio, mean NADH fluorescence lifetime and ratio of free to protein-bound NADH were used to quantify changes in metabolism. RESULTS: Results show that IFN-γ (M1) macrophages showed decreased optical redox ratios and mean NADH lifetime while phagocytosing immunogenic cancer cells compared to metastatic cells. To validate phagocytic function, a fluorescence microscopy-based protocol using a pH-sensitive fluorescent probe was used. Results indicate that M0 and M1 macrophages show similar trends in phagocytic potential. CONCLUSION: Overall, this work demonstrates that in vitro multiphoton imaging can be used to longitudinally track changes in phagocytosis and endogenous metabolic cofactors.

Autofluorescence imaging of endogenous metabolic cofactors in response to cytokine stimulation of classically activated macrophages.

BACKGROUND: Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates. METHODS: Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation. RESULTS: Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population. CONCLUSIONS: Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.

Generalized additive models to analyze nonlinear trends in biomedical longitudinal data using R: Beyond repeated measures ANOVA and linear mixed models.

In biomedical research, the outcome of longitudinal studies has been traditionally analyzed using the repeated measures analysis of variance (rm-ANOVA) or more recently, linear mixed models (LMEMs). Although LMEMs are less restrictive than rm-ANOVA as they can work with unbalanced data and non-constant correlation between observations, both methodologies assume a linear trend in the measured response. It is common in biomedical research that the true trend response is nonlinear and in these cases the linearity assumption of rm-ANOVA and LMEMs can lead to biased estimates and unreliable inference. In contrast, GAMs relax the linearity assumption of rm-ANOVA and LMEMs and allow the data to determine the fit of the model while also permitting incomplete observations and different correlation structures. Therefore, GAMs present an excellent choice to analyze longitudinal data with non-linear trends in the context of biomedical research. This paper summarizes the limitations of rm-ANOVA and LMEMs and uses simulated data to visually show how both methods produce biased estimates when used on data with non-linear trends. We present the basic theory of GAMs and using reported trends of oxygen saturation in tumors, we simulate example longitudinal data (2 treatment groups, 10 subjects per group, 5 repeated measures for each group) to demonstrate their implementation in R. We also show that GAMs are able to produce estimates with non-linear trends even when incomplete observations exist (with 40% of the simulated observations missing). To make this work reproducible, the code and data used in this paper are available at: https://github.com/aimundo/GAMs-biomedical-research.

Longitudinal examination of perfusion and angiogenesis markers in primary colorectal tumors shows distinct signatures for metronomic and maximum-tolerated dose strategies.

Metronomic chemotherapy (MET) has been developed to address the shortcomings of maximum-tolerated chemotherapy (MTD) in regard to toxicity and development of resistance mechanisms in the tumor. In colorectal cancer (CRC), MET is a promising novel strategy to treat locally advanced malignancies when used as neoadjuvant chemotherapy (NAC). However, so far there are no preclinical studies to assess the impact of MET NAC in CRC to assess the benefits and challenges of this approach. Here, we used a primary model of CRC (via azoxymethane) to analyze longitudinal changes in angiogenesis in primary tumors under MET and MTD NAC using a combination of diffuse reflectance spectroscopy and mRNA expression (via qPCR). Our results show that MET and MTD NAC lead to increased mean tissue oxygen saturation (8% and 5%, respectively) and oxyhemoglobin (15% and 10%) between weeks 2 and 5 of NAC, and that such increases are caused by distinct molecular signatures in the angiogenic program. Specifically, we find that in the MET group there is a sustained increase in Hif-1a, Aldoa, and Pgk1 expression, suggesting upregulated glycolysis, whereas MTD NAC causes a significant reduction in the expression of the aforementioned genes and of Vegf, leading to vascular remodeling in MTD-treated tumors. Taken together, this study demonstrates the ability of combined optical and molecular methodologies to provide a holistic picture of tumor response to therapy in CRC in a minimally invasive manner.

Macrophage-targeted anti-CCL2 immunotherapy enhances tumor sensitivity to 5-fluorouracil in a Balb/c-CT26 murine colon carcinoma model measured using diffuse reflectance spectroscopy.

BACKGROUND: Immunotherapy in colorectal cancer (CRC) regulates specific immune checkpoints and, when used in combination with chemotherapy, can improve patient prognosis. One specific immune checkpoint is the recruitment of circulating monocytes that differentiate into tumor-associated macrophages (TAMs) and promote tumor angiogenesis. Changes in vascularization can be non-invasively assessed via diffuse reflectance spectroscopy using hemoglobin concentrations and oxygenation in a localized tumor volume. In this study, we examine whether blockade of monocyte recruitment via CCL2 (macrophage chemoattractant protein-1) leads to enhanced sensitivity of 5-fluorouracil (5-FU) in a CT26-Balb/c mouse model of CRC. It was hypothesized that the blockade of TAMs will alter tumor perfusion, increasing chemotherapy response. A subcutaneous tumor model using Balb/c mice injected with CT26 colon carcinoma cells received either a saline or isotype control, anti-CCL2, 5-FU, or a combination of anti-CCL2 and 5-FU. RESULTS: Findings show that 12 days post-treatment, monocyte recruitment was significantly reduced by approximately 61% in the combination group. This shows that the addition of anti-CCL2 to 5-FU slowed the fold-change (change from the original measurement to the final measurement) in tumor volume from Day 0 to Day 12 (~ 5 fold). Modest improvements in oxygen saturation (~ 30%) were observed in the combination group. CONCLUSION: The findings in this work suggest that the blockade of CCL2 is sufficient in the reduction of TAMs that are recruited into the tumor microenvironment and has the ability to modestly alter tumor perfusion during early-tumor response to treatment even though the overall benefit is relatively modest.

Label-free optical biomarkers detect early calcific aortic valve disease in a wild-type mouse model.

BACKGROUND: Calcific aortic valve disease (CAVD) pathophysiology is a complex, multistage process, usually diagnosed at advanced stages after significant anatomical and hemodynamic changes in the valve. Early detection of disease progression is thus pivotal in the development of prevention and mitigation strategies. In this study, we developed a diet-based, non-genetically modified mouse model for early CAVD progression, and explored the utility of two-photon excited fluorescence (TPEF) microscopy for early detection of CAVD progression. TPEF imaging provides label-free, non-invasive, quantitative metrics with the potential to correlate with multiple stages of CAVD pathophysiology including calcium deposition, collagen remodeling and osteogenic differentiation. METHODS: Twenty-week old C57BL/6J mice were fed either a control or pro-calcific diet for 16 weeks and monitored via echocardiography, histology, immunohistochemistry, and quantitative polarized light imaging. Additionally, TPEF imaging was used to quantify tissue autofluorescence (A) at 755 nm, 810 nm and 860 nm excitation, to calculate TPEF 755-860 ratio (A860/525/(A755/460 + A860/525)) and TPEF Collagen-Calcium ratio (A810/525/(A810/460 + A810/525)) in the murine valves. In a separate experiment, animals were fed the above diets till 28 weeks to assess for later-stage calcification. RESULTS: Pro-calcific mice showed evidence of lipid deposition at 4 weeks and calcification at 16 weeks at the valve commissures. The valves of pro-calcific mice also showed positive expression for markers of osteogenic differentiation, myofibroblast activation, proliferation, inflammatory cytokines and collagen remodeling. Pro-calcific mice exhibited lower TPEF autofluorescence ratios, at locations coincident with calcification, that correlated with increased collagen disorganization and positive expression of osteogenic markers. Additionally, locations with lower TPEF autofluorescence ratios at 4 and 16 weeks exhibited increased calcification at later 28-week timepoints. CONCLUSIONS: This study suggests the potential of TPEF autofluorescence metrics to serve as a label-free tool for early detection and monitoring of CAVD pathophysiology.

Diffuse reflectance spectroscopy to monitor murine colorectal tumor progression and therapeutic response (Erratum).

The erratum notes a correction to Fig. 5 for the published article.

Diffuse reflectance spectroscopy to monitor murine colorectal tumor progression and therapeutic response.

SIGNIFICANCE: Many studies in colorectal cancer (CRC) use murine ectopic tumor models to determine response to treatment. However, these models do not replicate the tumor microenvironment of CRC. Physiological information of treatment response derived via diffuse reflectance spectroscopy (DRS) from murine primary CRC tumors provide a better understanding for the development of new drugs and dosing strategies in CRC. AIM: Tumor response to chemotherapy in a primary CRC model was quantified via DRS to extract total hemoglobin content (tHb), oxygen saturation (StO2), oxyhemoglobin, and deoxyhemoglobin in tissue. APPROACH: A multimodal DRS and imaging probe (0.78 mm outside diameter) was designed and validated to acquire diffuse spectra longitudinally-via endoscopic guidance-in developing colon tumors under 5-fluoruracil (5-FU) maximum-tolerated (MTD) and metronomic regimens. A filtering algorithm was developed to compensate for positional uncertainty in DRS measurements Results: A maximum increase in StO2 was observed in both MTD and metronomic chemotherapy-treated murine primary CRC tumors at week 4 of neoadjuvant chemotherapy, with 21 ± 6 % and 17 ± 6 % fold changes, respectively. No significant changes were observed in tHb. CONCLUSION: Our study demonstrates the feasibility of DRS to quantify response to treatment in primary CRC models.

Efficacy and clinical monitoring strategies for immune checkpoint inhibitors and targeted cytokine immunotherapy for locally advanced and metastatic colorectal cancer.

Colorectal cancer (CRC) is the fourth most common cancer type and is the second leading cause of cancer deaths annually in the United States. Conventional treatment options include postoperative (adjuvant) and preoperative (neoadjuvant) chemotherapy and radiotherapy. Although these treatment modalities have shown to decrease tumor burden, a major limitation to chemothearpy/radiotherapy is the high recurrence rate in patients. Immune-modulation strategies have emerged as a promising new therapeutic avenue to reduce this recurrence rate while minimizing undesirable systemic side effects. This review will focus specifically on the mechanisms of monoclonal antibodies: immune checkpoint inhibitors and cytokines, as well as current drugs approved by the Food and Drug Administration (FDA) and new clinical/pre-clinical trials. Finally, this review will investigate emerging methods used to monitor tumor response post-treatment.

Differences in colonic crypt morphology of spontaneous and colitis-associated murine models via second harmonic generation imaging to quantify colon cancer development.

BACKGROUND: Colorectal cancer remains the second leading cause of cancer death in the United States, and increased risk in patients with ulcerative colitis (a subset of inflammatory bowel disease) has motivated studies into early markers of dysplasia. The development of clinically translatable multiphoton imaging systems has allowed for the potential of in vivo label-free imaging of epithelial crypt structures via autofluorescence and/or second harmonic generation (SHG). SHG has been used to investigate collagen structures in various types of cancer, though the changes that colorectal epithelial collagen structures undergo during tumor development, specifically colitis-associated tumors, have not been fully investigated. METHODS: This study used two murine models, using A/J mice, one for spontaneous carcinoma and one for colitis-associated carcinoma, to investigate and quantify SHG image features that could potentially inform future study designs of endoscopic multiphoton imaging systems. The spontaneous tumor model comprised a series of six weekly injections of azoxymethane (AOM model). The colitis-associated tumor model comprised a single injection of AOM, followed by cycles of drinking water with dissolved dextran sodium sulfate salt (AOM-DSS model). SHG images of freshly resected murine colon were acquired with a multiphoton imaging system, and image features, such as crypt size, shape and distribution, were quantified using an automated algorithm. RESULTS: The comparison of quantified features of crypt morphology demonstrated the ability of our quantitative image feature algorithms to detect differences between spontaneous (AOM model) and colitis-associated (AOM-DSS model) murine colorectal tissue specimens. There were statistically significant differences in the mean and standard deviation of nearest neighbor (distance between crypts) and circularity between the Control cohort, AOM and AOM-DSS cohorts. We also saw significance between AOM and AOM-DSS cohorts when calculating nearest neighbor in images acquired at fixed depths. CONCLUSION: The results provide insight into the ability of SHG imaging to yield relevant data about the crypt microstructure in colorectal epithelium, specifically the potential to distinguish between spontaneous and colitis-associated murine models using quantification of crypt shape and distribution, informing future design of translational multiphoton imaging systems and protocols.

Effects of isoflurane anesthesia on physiological parameters in murine subcutaneous tumor allografts measured via diffuse reflectance spectroscopy.

Diffuse reflectance spectroscopy (DRS) has been used in murine studies to quantify tumor perfusion and therapeutic response. These studies frequently use inhaled isoflurane anesthesia, which depresses the respiration rate and results in the desaturation of arterial oxygen saturation, potentially affecting tissue physiological parameters. However, there have been no controlled studies quantifying the effect of isoflurane anesthesia on DRS-derived physiological parameters of murine tissue. The goal of this study was to perform DRS on Balb/c mouse (n = 10) tissue under various anesthesia conditions to quantify effects on tissue physiological parameters, including total hemoglobin concentration, tissue oxygen saturation, oxyhemoglobin and reduced scattering coefficient. Two independent variables were manipulated including metabolic gas type (pure oxygen vs. medical air) and isoflurane concentration (1.5 to 4.0%). The 1.5% isoflurane and 1 L/min oxygen condition most closely mimicked a no-anesthesia condition with oxyhemoglobin concentration within 89% ± 19% of control. The time-dependent effects of isoflurane anesthesia were tested, revealing that anesthetic induction with 4.0% isoflurane can affect DRS-derived physiological parameters up to 20 minutes post-induction. Finally, spectroscopy with and without isoflurane anesthesia was compared for colon tumor Balb/c-CT26 allografts (n = 5) as a representative model of subcutaneous murine tumor allografts. Overall, isoflurane anesthesia yielded experimentally-induced depressed oxyhemoglobin, and this depression was both concentration and time dependent. Investigators should understand the dynamic effects of isoflurane on tissue physiological parameters measured by DRS. These results may guide investigators in eliminating, limiting, or managing anesthesia-induced physiological changes in DRS studies in mouse models.

Sampling depth of a diffuse reflectance spectroscopy probe for in-vivo physiological quantification of murine subcutaneous tumor allografts.

Diffuse reflectance spectroscopy (DRS) is a probe-based spectral biopsy technique used in cancer studies to quantify tissue reduced scattering (µs') and absorption (µa) coefficients and vary in source-detector separation (SDS) to fine-tune sampling depth. In subcutaneous murine tumor allografts or xenografts, a key design requirement is ensuring that the source light interrogates past the skin layer into the tumor without significantly sacrificing signal-to-noise ratio (target of ≥15 dB). To resolve this requirement, a DRS probe was designed with four SDSs (0.75, 2.00, 3.00, and 4.00 mm) to interrogate increasing tissue volumes between 450 and 900 nm. The goal was to quantify percent errors in extracting µa and µs', and to quantify sampling depth into subcutaneous Balb/c-CT26 colon tumor allografts. Using an optical phantom-based experimental method, lookup-tables were constructed relating µa,µs', diffuse reflectance, and sampling depth. Percent errors were <10 % and 5% for extracting µa and µs', respectively, for all SDSs. Sampling depth reached up to 1.6 mm at the first Q-band of hemoglobin at 542 nm, the key spectral region for quantifying tissue oxyhemoglobin concentration. This work shows that the DRS probe can accurately extract optical properties and the resultant physiological parameters such as total hemoglobin concentration and tissue oxygen saturation, from sufficient depth within subcutaneous Balb/c-CT26 colon tumor allografts. Methods described here can be generalized for other murine tumor models. Future work will explore the feasibility of the DRS in quantifying volumetric tumor perfusion in response to anticancer therapies.

Two-layer inverse model for improved longitudinal preclinical tumor imaging in the spatial frequency domain.

Spatial frequency domain imaging (SFDI) is a widefield, noncontact, and label-free imaging modality that is currently being explored as a new tool for longitudinal tracking of cancer therapies in the preclinical setting. We describe a two-layer look-up-table (LUT) inversion algorithm for SFDI that better accounts for the skin (top layer) and tumor (bottom layer) tissue geometry in subcutaneous tumor models. Monte Carlo (MC) simulations were conducted natively in the spatial frequency domain, avoiding discretization errors associated with Fourier or Hankel transforms of conventional MC simulation results. The two-layer LUT was validated using two-layer tissue mimicking optical phantoms, in which the optical property extractions of the bottom (tumor) layer were determined to be within 20% and 11% of the true values for µa and µs', respectively. A sensitivity analysis was conducted to evaluate how imperfect top layer estimates affect bottom-layer optical property extractions. Finally, the two-layer LUT was used to reanalyze a prior longitudinal data set, which revealed larger therapy-induced changes in optical scattering and a more hypoxic tumor environment compared to the homogeneous LUT. The two-layer LUT described here improves the accuracy of subcutaneous tumor imaging, and the general methodology can be applied for arbitrary multilayer SFDI applications.

Optical redox ratio identifies metastatic potential-dependent changes in breast cancer cell metabolism.

The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Quantitative analysis of ex vivo colorectal epithelium using an automated feature extraction algorithm for microendoscopy image data.

Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia.

Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy.

Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns. The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.

In vivo measurement of non-keratinized squamous epithelium using a spectroscopic microendoscope with multiple source-detector separations.

In the non-keratinized epithelia, dysplasia typically arises near the basement membrane and proliferates into the upper epithelial layers over time. We present a non-invasive, multimodal technique combining high-resolution fluorescence imaging and broadband sub-diffuse reflectance spectroscopy (sDRS) to monitor health at various tissue layers. This manuscript focuses on characterization of the sDRS modality, which contains two source-detector separations (SDSs) of 374 µm and 730 µm, so that it can be used to extract in vivo optical parameters from human oral mucosa at two tissue thicknesses. First, we present empirical lookup tables (LUTs) describing the relationship between reduced scattering (µs') and absorption coefficients (µa) and absolute reflectance. LUTS were shown to extract µs' and µa with accuracies of approximately 4% and 8%, respectively. We then present LUTs describing the relationship between µs', µa and sampling depth. Sampling depths range between 210-480 and 260-620 µm for the 374 and 730 µm SDSs, respectively. We then demonstrate the ability to extract in vivo µs', µa, hemoglobin concentration, bulk tissue oxygen saturation, scattering exponent, and sampling depth from the inner lip of thirteen healthy volunteers to elucidate the differences in the extracted optical parameters from each SDS (374 and 730 µm) within non-keratinized squamous epithelia.

Towards monitoring dysplastic progression in the oral cavity using a hybrid fiber-bundle imaging and spectroscopy probe.

Intraepithelial dysplasia of the oral mucosa typically originates in the proliferative cell layer at the basement membrane and extends to the upper epithelial layers as the disease progresses. Detection of malignancies typically occurs upon visual inspection by non-specialists at a late-stage. In this manuscript, we validate a quantitative hybrid imaging and spectroscopy microendoscope to monitor dysplastic progression within the oral cavity microenvironment in a phantom and pre-clinical study. We use an empirical model to quantify optical properties and sampling depth from sub-diffuse reflectance spectra (450-750 nm) at two source-detector separations (374 and 730 µm). Average errors in recovering reduced scattering (5-26 cm(-1)) and absorption coefficients (0-10 cm(-1)) in hemoglobin-based phantoms were approximately 2% and 6%, respectively. Next, a 300 µm-thick phantom tumor model was used to validate the probe's ability to monitor progression of a proliferating optical heterogeneity. Finally, the technique was demonstrated on 13 healthy volunteers and volume-averaged optical coefficients, scattering exponent, hemoglobin concentration, oxygen saturation, and sampling depth are presented alongside a high-resolution microendoscopy image of oral mucosa from one volunteer. This multimodal microendoscopy approach encompasses both structural and spectroscopic reporters of perfusion within the tissue microenvironment and can potentially be used to monitor tumor response to therapy.