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Research Question: In a randomized, triple-blind, placebo-controlled clinical trial (RCT), we investigated the effect of astaxanthin (AST) on pro-inflammatory cytokines, oxidative stress (OS) markers, and assisted reproductive technology (ART) outcomes in 44 infertile Polycystic Ovary Syndrome (PCOS) patients., Design: Patients with PCOS were randomly divided into two groups. The intervention group received 6 mg AST, and the control group received placebo daily for 8 weeks. Blood samples were obtained from all patients before and after intervention and follicular fluid (FF) was collected during the ART procedure. Interleukin (IL) -6, IL-1β were evaluated from serum samples and FF and OS markers (malondialdehyde [MDA], catalase [CAT], superoxide dismutase [SOD], and reactive oxygen species [ROS]) were measured from FF. The groups were compared for ART outcomes as well., Results: A significant decrease in IL-6 and IL-1β concentrations (both, P = < 0.01) serum levels was found following AST treatment. FF cytokine levels and OS markers did not differ significantly between the groups. Reproductive outcomes, including the number of oocytes retrieved (P = 0.01), the MII oocyte count (P = 0.007), oocyte maturity rate (MII %) (P = 0.02) and number of frozen embryos (P = 0.03) significantly improved after intervention. No significant differences were found in chemical, clinical and multiple pregnancies between the groups., Conclusions: AST pretreatment may modify inflammation and improve ART outcomes in PCOS infertile patients. Further investigations are recommended to verify these findings., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: This study was designed to examine the effect of resveratrol on mitochondrial biogenesis, oxidative stress (OS), and assisted reproductive technology (ART) outcomes in individuals with polycystic ovary syndrome (PCOS)., Methods: Fifty-six patients with PCOS were randomly assigned to receive 800 mg/day of resveratrol or placebo for 60 days. The primary outcome was OS in follicular fluid (FF). The secondary outcome involved assessing gene and protein expression related to mitochondrial biogenesis, mitochondrial DNA (mtDNA) copy number, and adenosine triphosphate (ATP) content in granulosa cells (GCs). ART outcomes were evaluated at the end of the trial., Results: Resveratrol significantly reduced the total oxidant status (TOS) and oxidative stress index (OSI) in FF (P = 0.0142 and P = 0.0039, respectively) while increasing the total antioxidant capacity (TAC) (P < 0.0009). Resveratrol consumption also led to significant increases in the expression of critical genes involved in mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and mitochondrial transcription factor A (TFAM) (P = 0.0032 and P = 0.0003, respectively). However, the effect on nuclear respiratory factor 1 (Nrf-1) expression was not statistically significant (P = 0.0611). Resveratrol significantly affected sirtuin1 (SIRT1) and PGC-1α protein levels (P < 0.0001 and P = 0.0036, respectively). Resveratrol treatment improved the mtDNA copy number (P < 0.0001) and ATP content in GCs (P = 0.0014). Clinically, the resveratrol group exhibited higher rates of oocyte maturity (P = 0.0012) and high-quality embryos (P = 0.0013) than did the placebo group. There were no significant differences between the groups in terms of chemical or clinical pregnancy rates (P > 0.05)., Conclusions: These findings indicate that resveratrol may be a promising therapeutic agent for patients with PCOS undergoing assisted reproduction., Trial Registration Number: http://www.irct.ir ; IRCT20221106056417N1; 2023 February 09., (© 2024. The Author(s).)

Significance: Information about the spatial organization of fibers within a nerve is crucial to our understanding of nerve anatomy and its response to neuromodulation therapies. A serial block-face microscopy method [three-dimensional microscopy with ultraviolet surface excitation (3D-MUSE)] has been developed to image nerves over extended depths ex vivo . To routinely visualize and track nerve fibers in these datasets, a dedicated and customizable software tool is required., Aim: Our objective was to develop custom software that includes image processing and visualization methods to perform microscopic tractography along the length of a peripheral nerve sample., Approach: We modified common computer vision algorithms (optic flow and structure tensor) to track groups of peripheral nerve fibers along the length of the nerve. Interactive streamline visualization and manual editing tools are provided. Optionally, deep learning segmentation of fascicles (fiber bundles) can be applied to constrain the tracts from inadvertently crossing into the epineurium. As an example, we performed tractography on vagus and tibial nerve datasets and assessed accuracy by comparing the resulting nerve tracts with segmentations of fascicles as they split and merge with each other in the nerve sample stack., Results: We found that a normalized Dice overlap ( Dice norm ) metric had a mean value above 0.75 across several millimeters along the nerve. We also found that the tractograms were robust to changes in certain image properties (e.g., downsampling in-plane and out-of-plane), which resulted in only a 2% to 9% change to the mean Dice norm values. In a vagus nerve sample, tractography allowed us to readily identify that subsets of fibers from four distinct fascicles merge into a single fascicle as we move ∼ 5    mm along the nerve's length., Conclusions: Overall, we demonstrated the feasibility of performing automated microscopic tractography on 3D-MUSE datasets of peripheral nerves. The software should be applicable to other imaging approaches. The code is available at https://github.com/ckolluru/NerveTracker., (© 2024 The Authors.)

Context.—: Digital pathology using whole slide images has been recently approved to support primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining., Objective.—: To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations., Design.—: One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-μm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities., Results.—: The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard., Conclusions.—: Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis., Competing Interests: Borowsky, Levenson, and Fereidouni are cofounders of Histolix Inc; Gown is a consultant to Histolix Inc; and Morningstar was a paid full-time employee of Histolix Inc for part of the time of the study. Fleury has a financial interest in a capital group that has invested in Histolix. The other authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA., Competing Interests: M.L. was employed by the Foundation Medicine, Inc., R.L. is a co-founder of start-up MUSE Microscopy Inc. (now part of SmartPath Dx), and R.L., F.F. and A.B. are co-founders of start-up Histolix Inc. MUSE Microscopy Inc. and Histolix Inc both had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. The other authors declare no conflicts of interest.

Background: Prone positioning (PP) is a low-cost method with minimal risk to the patient that improves the oxygenation of patients with acute hypoxic respiratory failure (AHRF) due to COVID-19 pneumonia, thereby reducing their need for tracheal intubation (TI) and transferring to the intensive care unit (ICU). We aimed to overview the results of all previous systematic reviews and meta-analyses to examine the net effect of PP on oxygenation, the rate of TI and mortality in COVID-19 patients., Methods: We searched PubMed, Scopus, Web of Science, Google Scholar, and Cochrane Library databases from December 2019 through 2022 without publication language restriction for systematic reviews and meta-analysis studies on PP vs. supine position (SP) in conscious patients with hypoxic respiratory failure COVID-19. After study selection, data were extracted from published meta-analyses and pooled by comprehensive meta-analysis (CMA) software version 2.2.064 to achieve effect sizes. They were analyzed for TI and mortality rates dichotomous variables, and the results were shown as pooled odds ratios (OR) with a 95% confidence interval (CI). Continuous variables such as oxygenation indices (PaO2/FiO2 and SpO2) were also analyzed, and the data were shown as mean differences (MD) with lower and upper CI. The level of statistical significance was set at p ≤ 0.05., Results: Twelve systematic reviews and meta-analyses with 19,651 patients and six systematic reviews with 2,911 patients were included in this Review of Reviews (total: 22,562). PP treatment significantly reduced the rate of TI (OR = 0.639, %95 CI (0.492, 0.829); P-value = 0.001) and decreased mortality (OR = 0.363, %95 CI (0.240, 0.549), P-value < 0.001). There was no difference in PaO2/FiO2 (MD = 3.591[- 40.881, 48.062]; P-value = 0.874) and SpO2 percent (MD = 1.641[- 4.441, 7.723]; P-value = 0.597)., Conclusion: Prone positioning can be recommended in conscious ICU patients with COVID-19 pneumonia to reduce mortality and intubation., Systematic Review Registration: PROSPERO registration number: CRD42022326951. Registered 25 April 2022., (© 2024. The Author(s).)

Intraoperative identification of head and neck cancer tissue is essential to achieve complete tumor resection and mitigate tumor recurrence. Mesoscopic fluorescence lifetime imaging (FLIm) of intrinsic tissue fluorophores emission has demonstrated the potential to demarcate the extent of the tumor in patients undergoing surgical procedures of the oral cavity and the oropharynx. Here, we report FLIm-based classification methods using standard machine learning models that account for the diverse anatomical and biochemical composition across the head and neck anatomy to improve tumor region identification. Three anatomy-specific binary classification models were developed (i.e., "base of tongue," "palatine tonsil," and "oral tongue"). FLIm data from patients (N = 85) undergoing upper aerodigestive oncologic surgery were used to train and validate the classification models using a leave-one-patient-out cross-validation method. These models were evaluated for two classification tasks: (1) to discriminate between healthy and cancer tissue, and (2) to apply the binary classification model trained on healthy and cancer to discriminate dysplasia through transfer learning. This approach achieved superior classification performance compared to models that are anatomy-agnostic; specifically, a ROC-AUC of 0.94 was for the first task and 0.92 for the second. Furthermore, the model demonstrated detection of dysplasia, highlighting the generalization of the FLIm-based classifier. Current findings demonstrate that a classifier that accounts for tumor location can improve the ability to accurately identify surgical margins and underscore FLIm's potential as a tool for surgical guidance in head and neck cancer patients, including those subjects of robotic surgery.

Accurate quantification of renal fibrosis has profound importance in the assessment of chronic kidney disease (CKD). Visual analysis of a biopsy stained with trichrome under the microscope by a pathologist is the gold standard for evaluation of fibrosis. Trichrome helps to highlight collagen and ultimately interstitial fibrosis. However, trichrome stains are not always reproducible, can underestimate collagen content and are not sensitive to subtle fibrotic patterns. Using the Dual-mode emission and transmission (DUET) microscopy approach, it is possible to capture both brightfield and fluorescence images from the same area of a tissue stained with hematoxylin and eosin (H&E) enabling reproducible extraction of collagen with high sensitivity and specificity. Manual extraction of spectrally overlapping collagen signals from tubular epithelial cells and red blood cells is still an intensive task. We employed a UNet++ architecture for pixel-level segmentation and quantification of collagen using 760 whole slide image (WSI) patches from six cases of varying stages of fibrosis. Our trained model (Deep-DUET) used the supervised extracted collagen mask as ground truth and was able to predict the extent of collagen signal with a MSE of 0.05 in a holdout testing set while achieving an average AUC of 0.94 for predicting regions of collagen deposits. Expanding this work to the level of the WSI can greatly improve the ability of pathologists and machine learning (ML) tools to quantify the extent of renal fibrosis reproducibly and reliably.

Fluorescence imitating brightfield imaging (FIBI) is a novel microscopy method that allows for real-time, nondestructive, slide-free tissue imaging of fresh, formalin-fixed, or paraffin-embedded tissue. The nondestructive nature of the technology permits tissue preservation for downstream analyses. The objective of this observational study was to assess the utility of FIBI compared with conventional hematoxylin and eosin (H&E)-stained histology slides in feline gastrointestinal histopathology. Formalin-fixed paraffin-embedded full-thickness small intestinal tissue specimens from 50 cases of feline chronic enteropathy were evaluated. The ability of FIBI to evaluate predetermined morphological features (epithelium, villi, crypts, lacteals, fibrosis, submucosa, and muscularis propria) and inflammatory cells was assessed on a 3-point scale (0 = FIBI cannot identify the feature; 1 = FIBI can identify the feature; 2 = FIBI can identify the feature with more certainty than H&E). H&E and FIBI images were also scored according to World Small Animal Veterinary Association (WSAVA) Gastrointestinal Standardization Group guidelines. FIBI identified morphological features with similar or, in some cases, higher confidence compared with H&E images. The identification of inflammatory cells was less consistent. FIBI and H&E images showed an overall poor agreement with regard to the assigned WSAVA scores. While FIBI showed an equal or better ability to identify morphological features in intestinal biopsies, its ability to identify inflammatory cells is currently inferior compared with H&E-based imaging. Future studies on the utility of FIBI as a diagnostic tool for noninflammatory histopathologic lesions are warranted.

Background: Fluorescence imitating brightfield imaging (FIBI) is a novel alternative microscopy method that can image freshly excised, non-sectioned tissue. We examine its potential utility in dermatopathology by examining readily available specimens embedded in paraffin blocks., Methods: Nine skin samples embedded in paraffin blocks were superficially deparaffinized using xylene and ethanol and stained with H&E. FIBI captured tissue surface histopathology images using simple microscope optics and a color camera. We then applied deep-learning-based models to improve resemblance to standard H&E coloration and contrast. FIBI images were compared with corresponding standard H&E slides and concordance was assessed by two dermatopathologists who numerically scored epidermal and dermal structure appearance and overall diagnostic utility., Results: Dermatopathologist scores indicate that FIBI images are at least equivalent to standard H&E slides for visualizing structures such as epidermal layers, sweat glands, and nerves., Conclusion: Images acquired with FIBI are comparable to traditional H&E-stained slides, suggesting that this rapid, inexpensive, and non-destructive microscopy technique is a conceivable alternative to standard histopathology processes especially for time-sensitive procedures and in settings with limited histopathology resources., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Understanding peripheral nerve micro-anatomy can assist in the development of safe and effective neuromodulation devices. However, current approaches for imaging nerve morphology at the fiber level are either cumbersome, require substantial instrumentation, have a limited volume of view, or are limited in resolution/contrast. We present alternative methods based on MUSE (Microscopy with Ultraviolet Surface Excitation) imaging to investigate peripheral nerve morphology, both in 2D and 3D. For 2D imaging, fixed samples are imaged on a conventional MUSE system either label free (via auto-fluorescence) or after staining with fluorescent dyes. This method provides a simple and rapid technique to visualize myelinated nerve fibers at specific locations along the length of the nerve and perform measurements of fiber morphology (e.g., axon diameter and g-ratio). For 3D imaging, a whole-mount staining and MUSE block-face imaging method is developed that can be used to characterize peripheral nerve micro-anatomy and improve the accuracy of computational models in neuromodulation. Images of rat sciatic and human cadaver tibial nerves are presented, illustrating the applicability of the method in different preclinical models., (© 2022. The Author(s).)

Background: The application of radar systems in telecommunications and aerospace science is important. However, engineering department's staff various tissues are always under chronic radiation generated by the radar fields which may affect health., Objective: This study aims to evaluate the risk of radar wave exposure and to explore the effects and limitations., Material and Methods: In this simulation study, an adult body model versus 1 watt source with a distance of 50 centimeters exposure has been simulated using the CST STUDIO SUITE. Furthermore, various physical and electrical properties of each tissue and organ for different frequencies and exposure times have been studied. The exposure dose limitations have been considered using the International Commission on Non-Ionizing Radiation Protection (ICNIRP) safety and health guide report., Results: Total body absorbed doses for 4 GHz, 8 GHz, and 12 GHz frequency, and 6 min, 4 h, and 30 days exposure time, have been calculated as 1.136×10 -5 , 1.598×10 -5 , 1.58×10 -3 , 1.521×10 -5 , 3.122×10 -5 , 4.52×10 -3 , 4.1×10 -5 , 10 -4 , and 10 -2 , respectively., Conclusion: It has shown that the internal organs of the body and head will be under more risk by reducing radar frequencies from 12 GHz to 4 GHz. On the other hand, the higher frequency can cause a higher risk to the human skin. In addition, the maximum Specific Absorption Rate (SAR) for each case has been calculated. The results show that for this normalized source, the safety criteria have been respected, but for a higher source, the calculations must be repeated., (Copyright: © Journal of Biomedical Physics and Engineering.)

Minimally invasive core needle biopsies for medical diagnoses have become increasingly common for many diseases. Although tissue cores can yield more diagnostic information than fine needle biopsies and cytologic evaluations, there is no rapid assessment at the point-of-care for intact tissue cores that is low-cost and non-destructive to the biopsy. We have developed a proof-of-concept 3D printed millifluidic histopathology lab-on-a-chip device to automatically handle, process, and image fresh core needle biopsies. This device, named CoreView, includes modules for biopsy removal from the acquisition tool, transport, staining and rinsing, imaging, segmentation, and multiplexed storage. Reliable removal from side-cutting needles and bidirectional fluid transport of core needle biopsies of five tissue types has been demonstrated with 0.5 mm positioning accuracy. Automation is aided by a MATLAB-based biopsy tracking algorithm that can detect the location of tissue and air bubbles in the channels of the millifluidic chip. With current and emerging optical imaging technologies, CoreView can be used for a rapid adequacy test at the point-of-care for tissue identification as well as glomeruli counting in renal core needle biopsies.

Significance: 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is currently used for image-guided glioma resection. Typically, this widefield imaging method highlights the bulk of high-grade gliomas, but it underperforms at the infiltrating edge where PpIX fluorescence is not visible to the eyes. Fluorescence lifetime imaging (FLIm) has the potential to detect PpIX fluorescence below the visible detection threshold. Moreover, simultaneous acquisition of time-resolved nicotinamide adenine (phosphate) dinucleotide [NAD(P)H] fluorescence may provide metabolic information from the tumor environment to further improve overall tumor detection., Aim: We investigate the ability of pulse sampling, fiber-based FLIm to simultaneously image PpIX and NAD(P)H fluorescence of glioma infiltrative margins in patients., Approach: A mesoscopic fiber-based point-scanning FLIm device (355 nm pulses) was used to simultaneously resolve the fluorescence decay of PpIX (629/53 nm) and NAD(P)H (470/28 nm). The FLIm device enabled data acquisition at room light and rapid (<33  ms) augmentation of FLIm parameters on the surgical field-of-view. FLIm measurements from superficial tumors and tissue areas around the resection margins were performed on three glioblastoma patients in vivo following inspection of PpIX visible fluorescence with a conventional neurosurgical microscope. Microbiopsies were collected from FLIm imaged areas for histopathological evaluation., Results: The average lifetime from PpIX and NAD(P)H fluorescence distinguished between tumor and surrounding tissue. FLIm measurements of resection margins presented a range of PpIX and NAD(P)H lifetime values (τPpIX       ∼  3 to 14 ns, τNAD(P)H  =  3 to 6 ns) associated with unaffected tissue and areas of low-density tumor infiltration., Conclusions: Intraoperative FLIm could simultaneously detect the emission of PpIX and NAD(P)H from patients in vivo during craniotomy procedures. This approach doubles as a clinical tool to identify tumor areas while performing tissue resection and as a research tool to study tumor microenvironmental changes in vivo. Intraoperative FLIm of 5-ALA-induced PpIX and tissue autofluorescence makes a promising surgical adjunct to guide tumor resection surgery.

Smartphone microscopes can be useful tools for a broad range of imaging applications. This manuscript demonstrates the first practical implementation of Microscopy with Ultraviolet Surface Excitation (MUSE) in a compact smartphone microscope called Pocket MUSE, resulting in a remarkably effective design. Fabricated with parts from consumer electronics that are readily available at low cost, the small optical module attaches directly over the rear lens in a smartphone. It enables high-quality multichannel fluorescence microscopy with submicron resolution over a 10× equivalent field of view. In addition to the novel optical configuration, Pocket MUSE is compatible with a series of simple, portable, and user-friendly sample preparation strategies that can be directly implemented for various microscopy applications for point-of-care diagnostics, at-home health monitoring, plant biology, STEM education, environmental studies, etc.

Vagus nerve stimulation (VNS) is a method to treat drug-resistant epilepsy and depression, but therapeutic outcomes are often not ideal. Newer electrode designs such as intra-fascicular electrodes offer potential improvements in reducing off-target effects but require a detailed understanding of the fascicular anatomy of the vagus nerve. We have adapted a section-and-image technique, cryo-imaging, with UV excitation to visualize fascicles along the length of the vagus nerve. In addition to offering optical sectioning at the surface via reduced penetration depth, UV illumination also produces sufficient contrast between fascicular structures and connective tissue. Here we demonstrate the utility of this approach in pilot experiments. We imaged fixed, cadaver vagus nerve samples, segmented fascicles, and demonstrated 3D tracking of fascicles. Such data can serve as input for computer models of vagus nerve stimulation.

Multiple sclerosis (MS) is presented as the most common autoimmune and demyelinating neurological disorder with incapacitating complications and with no definite therapy. Most treatments for MS mainly focus on attenuation of its severity and recurrence. To model MS reliably to study pathogenesis and efficacy of possible chemicals, experimental autoimmune encephalomyelitis (EAE) condition is induced in rodents. Ellagic acid is a neuroprotective polyphenol that can protect against demyelination. This study was planned and conducted to assess its possible beneficial effect in MOG-induced EAE model of MS with emphasis on uncovering its modes of action. Ellagic acid was given p.o. (at doses of 10 or 50 mg/kg/day) after development of clinical signs of MS to C57BL/6 mice immunized with MOG 35-55 . Results showed that ellagic acid can ameliorate severity of the disease and partially restore tissue level of TNFα, IL-6, IL-17A and IL-10. Besides, ellagic acid lowered tissue levels of NLRP3 and caspase 1 in addition to its mitigation of neuroinflammation, demyelination and axonal damage in spinal cord specimens of EAE group. As well, ellagic acid treatment prevented reduction of MBP and decreased GFAP and Iba1 immunoreactivity. Taken together, ellagic acid can decrease severity of EAE via amelioration of astrogliosis, astrocyte activation, demyelination, neuroinflammation and axonal damage that is partly related to its effects on NLRP3 inflammasome and pyroptotic pathway., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the clinical practice of pathology, trichrome stains are commonly used to highlight collagen and to help evaluate fibrosis. Such stains do delineate collagen deposits but are not molecularly specific and can suffer from staining inconsistencies. Moreover, performing histochemical stain evaluation requires the preparation of additional sections beyond the original hematoxylin- and eosin-stained slides, as well as additional staining steps, which together add cost, time, and workflow complications. We have developed a new microscopy approach, termed DUET (DUal-mode Emission and Transmission) that can be used to extract signals that would typically require special stains or advanced optical methods. Our preliminary analysis demonstrates the potential of using the resulting signals to generate virtual histochemical images that resemble trichrome-stained slides and can support clinical evaluation. We demonstrate advantages of this approach over images acquired from conventional trichrome-stained slides and compare them with images created using second harmonic generation microscopy., Competing Interests: FF: HistoliX (I,P) RL: HistoliX (I,P), (© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Traditional histology relies on processing and physically sectioning either frozen or formalin-fixed paraffin-embedded (FFPE) tissue into thin slices (typically 4-6 μm) prior to staining and viewing on a standard wide-field microscope. Microscopy using ultraviolet (UV) surface excitation (MUSE) represents a novel alternative microscopy method that works with UV excitation using oblique cis-illumination, which can generate high-quality images from the cut surface of fresh or fixed tissue after brief staining, with no requirement for fixation, embedding and histological sectioning of tissue specimens. We examined its potential utility in dermatopathology. Concordance between MUSE images and hematoxylin and eosin (H&E) slides was assessed by the scoring of MUSE images on their suitability for identifying 10 selected epidermal and dermal structures obtained from minimally fixed tissue, including stratum corneum, stratum granulosum, stratum spinosum, stratum basale, nerve, vasculature, collagen and elastin, sweat glands, adipose tissue and inflammatory cells, as well as 4 cases of basal cell carcinoma and 1 case of pseudoxanthoma elasticum deparaffinized out of histology blocks. Our results indicate that MUSE can identify nearly all normal skin structures seen on routine H&E as well as some histopathologic features, and appears promising as a fast, reliable and cost-effective diagnostic approach in dermatopathology., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Multispectral imaging (MSI) is increasingly finding application in the study and characterization of biological specimens. However, the methods typically used come with challenges on both the acquisition and the analysis front. MSI can be slow and photon-inefficient, leading to long imaging times and possible phototoxicity and photobleaching. The resulting datasets can be large and complex, prompting the development of a number of mathematical approaches for segmentation and signal unmixing. We show that under certain circumstances, just three spectral channels provided by standard color cameras, coupled with multispectral analysis tools, including a more recent spectral phasor approach, can efficiently provide useful insights. These findings are supported with a mathematical model relating spectral bandwidth and spectral channel number to achievable spectral accuracy. The utility of 3-band RGB and MSI analysis tools are demonstrated on images acquired using brightfield and fluorescence techniques, as well as a novel microscopy approach employing UV-surface excitation. Supervised linear unmixing, automated non-negative matrix factorization and phasor analysis tools all provide useful results, with phasors generating particularly helpful spectral display plots for sample exploration.

Histological examination of tissues is central to the diagnosis and management of neoplasms and many other diseases and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides-a process that can require hours or days, contributes to cost and delays access to critical information. Here, we introduce a simple, non-destructive slide-free technique that, within minutes, provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin and eosin histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280 nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA sequencing). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and low-resource settings and to provide opportunities for rapid histology in research.

Fluorescence lifetime imaging has been shown to serve as a valuable tool for interrogating and diagnosis of biological tissue at a mesoscopic level. The ability to analyze fluorescence decay curves to extract lifetime values in real-time is crucial for clinical translation and applications such as tumor margin delineation or intracoronary imaging of atherosclerotic plaques. In this work, we compare the performance of two popular non-parametric (fit-free) methods for determining lifetime values from fluorescence decays in real-time-the Phasor approach and Laguerre deconvolution. We demonstrate results from simulated and experimental data to compare the accuracy and speed of both methods and their dependence on noise and model parameters.

Background: We wanted to investigate the physical state of biological membranes in live cells under the most physiological conditions possible. Methods: For this we have been using laurdan, C-laurdan or M-laurdan to label a variety of cells, and a biphoton microscope equipped with both a thermostatic chamber and a spectral analyser. We also used a flow cytometer to quantify the 450/530 nm ratio of fluorescence emissions by whole cells. Results: We find that using all the information provided by spectral analysis to perform spectral decomposition dramatically improves the imaging resolution compared to using just two channels, as commonly used to calculate generalized polarisation (GP). Coupled to a new plugin called Fraction Mapper, developed to represent the fraction of light intensity in the first component in a stack of two images, we obtain very clear pictures of both the intra-cellular distribution of the probes, and the polarity of the cellular environments where the lipid probes are localised. Our results lead us to conclude that, in live cells kept at 37°C, laurdan, and M-laurdan to a lesser extent, have a strong tendency to accumulate in the very apolar environment of intra-cytoplasmic lipid droplets, but label the plasma membrane (PM) of mammalian cells ineffectively. On the other hand, C-laurdan labels the PM very quickly and effectively, and does not detectably accumulate in lipid droplets. Conclusions: From using these probes on a variety of mammalian cell lines, as well as on cells from Drosophila and Dictyostelium discoideum , we conclude that, apart from the lipid droplets, which are very apolar, probes in intracellular membranes reveal a relatively polar and hydrated environment, suggesting a very marked dominance of liquid disordered states. PMs, on the other hand, are much more apolar, suggesting a strong dominance of liquid ordered state, which fits with their high sterol contents., Competing Interests: Competing interests: No competing interests were disclosed.

Quantitative magnetic resonance imaging (qMRI) is a versatile, non-destructive and non-invasive tool in life, material, and medical sciences. When multiple components contribute to the signal in a single pixel, however, it is difficult to quantify their individual contributions and characteristic parameters. Here we introduce the concept of phasor representation to qMRI to disentangle the signals from multiple components in imaging data. Plotting the phasors allowed for decomposition, unmixing, segmentation and quantification of our in vivo data from a plant stem, a human and mouse brain and a human prostate. In human brain images, we could identify 3 main T 2 components and 3 apparent diffusion coefficients; in human prostate 5 main contributing spectral shapes were distinguished. The presented phasor analysis is model-free, fast and accurate. Moreover, we also show that it works for undersampled data.

Collagen and other components in the extracellular matrix are proving of increasing importance for the understanding of complex cell and tissue interactions in a variety of settings. Detection and quantitation of these components can still prove challenging, and a number of techniques have been developed. We focus here on methods in fluorescence-based assessments, including multiplexed immunodetection and the use of simpler histochemical stains, both complemented by linear unmixing techniques. Typically, differentiating these components requires the use of a set of optical filters to isolate each fluorescent compound from each other and from often bright background autofluorescence signals. However, standard fluorescent microscopes are usually only able to separate a limited number of components. If the emission spectra of the fluorophores are spectrally distinct, but overlapping, sophisticated spectral imaging or computational methods can be used to optimize separation and quantitation. This chapter describes spectral unmixing methodology and associated open-source software tools available to analyze multispectral as well as simple color (RGB) images.

Background: Skin care products make up the largest part (36%) of the cosmetic market globally, of which the United States plays the largest role. In 2015, approximately 115 billion USD was spent globally on skin care products. Skin care products, in contradistinction to pharmaceuticals, are not strictly regulated by the FDA. A key factor for evaluation of a skin care product or topical drug is skin barrier function and effect on super cial skin. Thus, it is critical to have quantitative and qualitative methods to study the effects of skin care products on skin barrier and the super cial skin. Currently, no imaging method exists that can evaluate and track super cial skin changes visually in real-time., Objective: To report using a novel imaging modality, Microscopy using Ultraviolet Surface Excitation (MUSE), to provide real-time, high- resolution, in vivo characterization of super cial skin and moisturizing properties of topical moisturizer, and to highlight key bene ts of using MUSE to visualize the super cial skin and serve as an excellent complementary tool to current quantitative methods., Methods and Materials: The methodology of MUSE is based upon two main principles inherent to ultraviolet (UV) light and uorescent staining agents. In this study, the author's (JJ) index ngertip was imaged using the MUSE instrument without and with moisturizer., Results: Dermatoglyphics of the fingertip consists of ridges (cristae super ciales) and grooves (sulci super ciales) proved to be straightforward to visualize at high resolution. Desquamation of superficial corneocytes and opening of an acrosyringium (the most superficial portion of eccrine ducts) were visualized in high-resolution. Post-application of a moisturizer, a uniform layer of moisturizer could be seen superficial to the corneocytes along the ridges and CONCLUSIONS: Real-time, high-resolution, in vivo characterization of super cial skin and moisturizing properties of moisturizer using MUSE is feasible. Its utility can be enhanced with downstream quantification using imaging software. J Drugs Dermatol. 2016;15(11):1344-1346..

Sjögren's syndrome (SS) is a debilitating autoimmune disease. Patients with SS may develop xerostomia. This process is progressive, and there are no therapeutics that target disease etiology. We hypothesized BAFF receptor (BAFFR) blockade would mitigate SS disease development, and neutralization of CXCL13 and BAFF signaling would be more efficacious than BAFFR blockade alone. We treated NOD/ShiLtJ SS mice with soluble BAFF receptor (BAFFR-Fc) or anti-CXCL13/BAFFR-Fc in combination, prior to the development of clinical disease. Our results show treatment with BAFFR-Fc reduced peripheral B cell numbers and decreased sialadenitis. In addition, this treatment reduced total serum immunoglobulin as well as IgG and IgM specific anti-nuclear autoantibodies. NOD/ShiLtJ mice treated with BAFFR-Fc and anti-CXCL13 antibody were protected from salivary deficits. Results from this study suggest blockade of CXCL13 and BAFFR together may be an effective therapeutic strategy in preventing salivary hypofunction and reducing autoantibody titers and sialadenitis in patients with SS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: The purpose of the present study was to examine the cognitive, emotional and behavioral differences between obese and normal weight women., Methods: The participants consisted of 60 obese women (BMI ≥ 35, Mean age 35.83) who were candidates for bariatric surgery and 60 normal weight women (BMI ≤ 24.90, Mean age 33.38) who were selected through convenient sampling method. The two groups were matched with respect to age and education. Measures included the Young Schema Questionnaire-Short Form, Difficulties in Emotion Regulation Scale (DERS) and the Binge Eating Scale. Independent sample t test and Mann-Whitney U tests were conducted to compare the two groups' scores on early maladaptive schemas, DER and binge eating., Results: Results indicated that obese women candidate for bariatric surgery scored significantly higher on emotional deprivation, mistrust, failure, dependency, enmeshment, self-sacrifice, especially abandonment, social isolation, vulnerability, self-control and subjugation schemas, but not on shame, entitlement, emotional inhibition and unrelenting standards. Obese women also displayed higher scores on three subscales of DERS, i.e., goal, impulse and strategies. BE scores were significantly higher in obese women than normal weight ones., Conclusion: The findings showed that obese women candidates for bariatric surgery suffered from more cognitive, emotional and behavioral vulnerability compared to women with normal weight. Addressing these vulnerabilities among obese women could improve outcomes of weight loss surgeries and cognitive behavioral interventions so that weight regain is minimized and better outcomes are achieved.

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The combined analysis of spectral and lifetime images has the potential to provide more accurate and more detailed information about Förster resonance energy transfer (FRET). We have developed a novel FRET analysis method to analyze images recorded by multispectral lifetime imaging. The new method is based on a phasor approach and facilitates the simultaneous analysis of decay kinetics of donor and acceptor molecules. The method is applicable to both molecules that exhibit a mono-exponential decay and a bi-exponential decay. As an example we show the possibility of extracting the energy transfer efficiency and the fraction of interacting molecules even in the presence of non-interacting molecules. The reliability of the method is investigated by comparing it with conventional FRET-FLIM analyses. We show that, with the same number of detected photons, the spectrally resolved phasor approach provides higher accuracy than other analysis methods; the confidence interval is improved and the FRET efficiency is closer to the real value.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

A method, is presented for blind unmixing spectrally resolved fluorescence lifetime images. The method is based on the combined analysis of spectral and lifetime phasors and allows unmixing of up to three components without any prior knowledge. Fractional intensities, spectra and decay curves of the individual components can be extracted with this new technique. The reliability and sensitivity are investigated and the possibility of extending the method to unmix more components is discussed. The method is evaluated on mixtures of fluorescent dyes and labeled cells.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A new global analysis algorithm to analyse (hyper-) spectral images is presented. It is based on the phasor representation that has been demonstrated to be very powerful for the analysis of lifetime imaging data. In spectral phasor analysis the fluorescence spectrum of each pixel in the image is Fourier transformed. Next, the real and imaginary components of the first harmonic of the transform are employed as X and Y coordinates in a scatter (spectral phasor) plot. Importantly, the spectral phasor representation allows for rapid (real time) semi-blind spectral unmixing of up to three components in the image. This is demonstrated on slides with fixed cells containing three fluorescent labels. In addition the method is used to analyse autofluorescence of cells in a fresh grass blade. It is shown that the spectral phasor approach is compatible with spectral imaging data recorded with a low number of spectral channels.