NerveTracker: a Python-based software toolkit for visualizing and tracking groups of nerve fibers in serial block-face microscopy with ultraviolet surface excitation images.

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.

Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

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.

A Pilot Validation Study Comparing Fluorescence-Imitating Brightfield Imaging, A Slide-Free Imaging Method, With Standard Formalin-Fixed, Paraffin-Embedded Hematoxylin-Eosin-Stained Tissue Section Histology for Primary Surgical Pathology Diagnosis.

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.

Improving quantification of renal fibrosis using Deep-DUET.

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.

Anatomy-Specific Classification Model Using Label-Free FLIm to Aid Intraoperative Surgical Guidance of Head and Neck Cancer.

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.

Utility of fluorescence imitating brightfield imaging microscopy for the diagnosis of feline chronic enteropathy.

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.

Pilot study of fluorescence imitating brightfield imaging for rapid, slide-free dermatopathology.

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.

Imaging peripheral nerve micro-anatomy with MUSE, 2D and 3D approaches.

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.

CoreView: fresh tissue biopsy assessment at the bedside using a millifluidic imaging chip.

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.

First in patient assessment of brain tumor infiltrative margins using simultaneous time-resolved measurements of 5-ALA-induced PpIX fluorescence and tissue autofluorescence.

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.

Pocket MUSE: an affordable, versatile and high-performance fluorescence microscope using a smartphone.

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.

3D imaging of the vagus nerve fascicular anatomy with cryo-imaging and UV excitation.

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.

Dual-mode emission and transmission microscopy for virtual histochemistry using hematoxylin- and eosin-stained tissue sections.

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.

Microscopy with ultraviolet surface excitation (MUSE): A novel approach to real-time inexpensive slide-free dermatopathology.

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.

Multispectral analysis tools can increase utility of RGB color images in histology.

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.

Spectral Unmixing Methods and Tools for the Detection and Quantitation of Collagen and Other Macromolecules in Tissue Specimens.

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.

Using spectral decomposition of the signals from laurdan-derived probes to evaluate the physical state of membranes in live cells.

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.

Rapid fluorescence lifetime estimation with modified phasor approach and Laguerre deconvolution: a comparative study.

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.

Multi-component quantitative magnetic resonance imaging by phasor representation.

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.