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 . To routinely visualize and track nerve fibers in these datasets, a dedicated and customizable software tool is required.

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.

Improving quantification of renal fibrosis using .

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Microscopy with ultraviolet surface excitation for rapid slide-free histology.

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.

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.

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

We wanted to investigate the physical state of biological membranes in live cells under the most physiological conditions possible. 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.

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.

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.

Real-time, High-resolution, In Vivo Characterization of Superficial Skin With Microscopy Using Ultraviolet Surface Excitation (MUSE).

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.

Early BAFF receptor blockade mitigates murine Sjögren's syndrome: Concomitant targeting of CXCL13 and the BAFF receptor prevents salivary hypofunction.

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.

Phasor based analysis of FRET images recorded using spectrally resolved lifetime imaging.

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.

Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif.

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).