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.

Minimally invasive in vivo endoscopic monitoring of dextran sulfate sodium-induced murine colitis.

Ulcerative colitis (UC) is a gastrointestinal, autoimmune disease that causes ulceration and inflammation of the colon with an incidence of 10 out of every 100,000 people in North America and Western Europe. Though the specific cause is unknown, several studies have demonstrated that inflammatory cells as well as environmental variables, genetics, and lifestyle behaviors can play a role in the long-term inflammatory response. Researchers have commonly used immunohistochemistry, western blotting and gene sequencing to establish the cellular processes behind UC relapse and remission. However, because these destructive methods necessitate the removal of a sample, they can only be used on non-living tissues. The use of minimally invasive approaches to evaluate the in vivo, longitudinal effects of UC on the mucosa in the colon is gaining popularity among clinicians and researchers. We have created a dextran sulfate sodium-induced model of UC in C57 mice based on the work of Wirtz et al., and a minimally invasive imaging modality to explore the changes in mucosal tissue during "active" and "in remission" UC. Briefly, C57 mice were given dextran sulfate sodium (DSS) dissolved in water in 5-day cycles with a remission/recovery period of 10 days. After 7 days post-DSS treatment and 7 days post-recovery, mice were anesthetized and exploratory endoscopies were performed to assess the mucosal changes that occur during the "active" and "remission" periods of UC. Value of protocol:•Minimally invasive induction of ulcerative colitis in a murine mouse model.•Minimally invasive longitudinal monitoring of "active" and "in remission" ulcerative colitis.•Our endoscopic based imaging modality can be used to validate the induction of ulcerative colitis and the potential treatment response for pre-clinical trials.

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.

Employing Faculty, Peer Mentoring, and Coaching to Increase the Self-Confidence and Belongingness of First-Generation College Students in Biomedical Engineering.

First-generation college students (FGCSs) face myriad challenges including the lack of parental guidance, economic and social burdens, isolation, decreased belongingness, and lowered self-confidence making them at an increased risk of dropping out of college compared to their continuing-generation college students colleagues. In addition, being in a multidisciplinary science, technology, engineering, and mathematics (STEM) field such as biomedical engineering (BMEG) is another challenge as it requires the integration of several disciplines. This study aims to maximize FGCSs' success and retention in BMEG. We hypothesize that STEM-tailored faculty and peer mentoring that is focused on academic and professional development will significantly increase BMEG FGCSs' academic and professional success and enhance their belongingness to the engineering community. Study participants were assigned to either group; faculty mentoring combined with academic coaching or peer mentoring combined with academic coaching. Both quantitative and qualitative data were collected using two surveys: prementoring and postmentoring. Both faculty mentoring and peer mentoring led to increasing FGCSs' confidence, belongingness, and involvement in professional opportunities. To tackle the added challenge of studying a multidisciplinary STEM field to the challenges facing FGCSs, a mentorship program that is focused on enhancing self-confidence, sense of belonging and augmenting professional development can be employed to ensure the success, integration, and retention of FGCSs in multidisciplinary STEM fields such as BMEG.

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.

A Light-Sheet-Based Imaaging Spectrometer to Characterize Acridine Orange Fluorescence within Leukocytes.

Low-cost imaging systems that utilize exogenous fluorescent dyes, such as acridine orange (AO), have recently been developed for use as point-of-care (POC) blood analyzers. AO-based fluorescence imaging exploits variations in emission wavelength within different cell types to enumerate and classify leukocyte subpopulations from whole blood specimens. This approach to leukocyte classification relies on accurate and reproducible colorimetric features, which have previously been demonstrated to be highly dependent on the cell staining protocols (such as specific AO concentration, timing, and pH). We have developed a light-sheet-based fluorescence imaging spectrometer, featuring a spectral resolution of 9 nm, with an automated spectral extraction algorithm as an investigative tool to study the spectral features from AO-stained leukocytes. Whole blood specimens were collected from human subjects, stained with AO using POC methods, and leukocyte spectra were acquired on a cell-by-cell basis. The post-processing method involves three steps: image segmentation to isolate individual cells in each spectral image; image quality control to exclude cells with low emission intensity, out-of-focus cells, and cellular debris; and the extraction of spectra for each cell. An increase in AO concentration was determined to contribute to the red-shift in AO-fluorescence, while varied pH values did not cause a change in fluorescence. In relation to the spectra of AO-stained leukocytes, there were corresponding red-shift trends associated with dye accumulation within acidic vesicles and at increasing incubation periods. The system presented here could guide future development of POC systems reliant on AO fluorescence and colorimetric features to identify leukocyte subpopulations in whole blood specimens.

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.

Evaluation of Acridine Orange Staining for a Semi-Automated Urinalysis Microscopic Examination at the Point-of-Care.

A urinary tract infection (UTI) can be diagnosed via urinalysis, consisting of a dipstick test and manual microscopic examination. Point-of-care (POC) image-based systems have been designed to automate the microscopic examination for low-volume laboratories or low-resource clinics. In this pilot study, acridine orange (AO) was evaluated as a fluorescence-based contrast agent to aid in detecting and enumerating urine sediment specific for diagnosing a UTI. Acridine orange staining of epithelial cells, leukocytes, and bacteria provided sufficient contrast to successfully implement image segmentation techniques, which enabled the extraction of classifiable morphologic features. Surface area bounded by each cell border was used to differentiate the sediment; epithelial cells were larger than 500µm2, bacteria were less than 30µm2, and leukocytes in between. This image-based semi-automated technique using AO resulted in similar cell counts to the clinical results, which demonstrates the feasibility of AO as an aid for POC urinalysis systems.

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.

Redox-Magnetohydrodynamically Controlled Fluid Flow with Poly(3,4-ethylenedioxythiophene) Coupled to an Epitaxial Light Sheet Confocal Microscope for Image Cytometry Applications.

We present the merging of two technologies to perform continuous high-resolution fluorescence imaging of cellular suspensions in a deep microfluidics chamber with no moving parts. An epitaxial light sheet confocal microscope (e-LSCM) was used to image suspensions enabled by fluid transport via redox-magnetohydrodynamics (R-MHD). The e-LSCM features a linear solid state sensor, oriented perpendicular to the direction of flow, that can bin the emission across different numbers of pixels, yielding electronically adjustable optical sectioning. This, in addition to intensity thresholding, defines the axial resolution, which was validated with an optical phantom of polystyrene microspheres suspended in agarose. The linear fluid speed within the microfluidics chamber was uniform (0.16-2.9%) across the 0.5-1.0 mm lateral field of view (dependent upon the chosen magnification) with continuous acquisition. Also, the camera's linear exposure periods were controlled to ensure an accurate image aspect ratio across this span. Poly(3,4-ethylenedioxythiophene) (PEDOT) was electrodeposited as an immobilized redox film on electrodes of a chip for R-MHD, and the fluid flow was calibrated to specific linear speeds as a function of applied current. Images of leukocytes stained with acridine orange, a fluorescent, amphipathic vital dye that intercalates DNA, were acquired in the R-MHD microfluidics chamber with the e-LSCM to demonstrate imaging of biological samples. The combination of these technologies provides a miniaturizable platform for large sample volumes and high-throughput, image-based analysis without the requirement of moving parts, enabling development of robust, point-of-care image cytometry.

Fluorescein as a topical fluorescent contrast agent for quantitative microendoscopic inspection of colorectal epithelium.

Fiber bundle microendoscopic imaging of colorectal tissue has shown promising results, for both qualitative and quantitative analysis. A quantitative image quality control and image feature extraction algorithm was previously designed for quantitative image feature analysis of proflavine-stained ex vivo colorectal tissue. We investigated fluorescein as an alternative topical stain. Images of ex vivo porcine, caprine, and human colorectal tissue were used to compare microendoscopic images of tissue topically stained with fluorescein and proflavine solutions. Fluorescein was shown to be comparable for automated crypt detection, with an average crypt detection sensitivity exceeding 90% using a combination of three contrast limit pairs.