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.

Simultaneous deep ultraviolet transmission and scattering microscopy for virtual histology.

In recent years, the emergence of a variety of novel optical microscopy techniques has enabled the generation of virtual optical stains of unlabeled tissue specimens, which have the potential to transform existing clinical histopathology workflows. In this work, we present a simultaneous deep ultraviolet transmission and scattering microscopy system that can produce virtual histology images that show concordance to conventional gold-standard histological processing techniques. The results of this work demonstrate the system's diagnostic potential for characterizing unlabeled thin tissue sections and streamlining histological workflows.

Deep ultraviolet fluorescence microscopy of three-dimensional structures in the mouse brain.

Three-dimensional (3D) imaging at cellular resolution improves our understanding of the brain architecture and is crucial for structural and functional integration as well as for the understanding of normal and pathological conditions in the brain. We developed a wide-field fluorescent microscope for 3D imaging of the brain structures using deep ultraviolet (DUV) light. This microscope allowed fluorescence imaging with optical sectioning due to the large absorption at the surface of the tissue and hence low tissue penetration of DUV light. Multiple channels of fluorophore signals were detected using single or a combination of dyes emitting fluorescence in the visible range of spectrum upon DUV excitation. Combination of this DUV microscope with microcontroller-based motorized stage enabled wide-field imaging of a coronal section of the cerebral hemisphere in mouse for deciphering cytoarchitecture of each substructure in detail. We extended this by integrating vibrating microtome which allowed serial block-face imaging of the brain structure such as the habenula in mouse. Acquired images were with resolution high enough for quantification of the cell numbers and density in the mouse habenula. Upon block-face imaging of the tissues covering entire extent of the cerebral hemisphere of the mouse brain, acquired data were registered and segmented for quantification of cell number in each brain regions. Results in the current analysis indicated that this novel microscope could be a convenient tool for large-scale 3D analysis of the brain in mice.

Label-free intraoperative histology of bone tissue via deep-learning-assisted ultraviolet photoacoustic microscopy.

Obtaining frozen sections of bone tissue for intraoperative examination is challenging. To identify the bony edge of resection, orthopaedic oncologists therefore rely on pre-operative X-ray computed tomography or magnetic resonance imaging. However, these techniques do not allow for accurate diagnosis or for intraoperative confirmation of the tumour margins, and in bony sarcomas, they can lead to bone margins up to 10-fold wider (1,000-fold volumetrically) than necessary. Here, we show that real-time three-dimensional contour-scanning of tissue via ultraviolet photoacoustic microscopy in reflection mode can be used to intraoperatively evaluate undecalcified and decalcified thick bone specimens, without the need for tissue sectioning. We validate the technique with gold-standard haematoxylin-and-eosin histology images acquired via a traditional optical microscope, and also show that an unsupervised generative adversarial network can virtually stain the ultraviolet-photoacoustic-microscopy images, allowing pathologists to readily identify cancerous features. Label-free and slide-free histology via ultraviolet photoacoustic microscopy may allow for rapid diagnoses of bone-tissue pathologies and aid the intraoperative determination of tumour margins.

Label-free imaging and classification of live P. falciparum enables high performance parasitemia quantification without fixation or staining.

Manual microscopic inspection of fixed and stained blood smears has remained the gold standard for Plasmodium parasitemia analysis for over a century. Unfortunately, smear preparation consumes time and reagents, while manual microscopy is skill-dependent and labor-intensive. Here, we demonstrate that deep learning enables both life stage classification and accurate parasitemia quantification of ordinary brightfield microscopy images of live, unstained red blood cells. We tested our method using both a standard light microscope equipped with visible and near-ultraviolet (UV) illumination, and a custom-built microscope employing deep-UV illumination. While using deep-UV light achieved an overall four-category classification of Plasmodium falciparum blood stages of greater than 99% and a recall of 89.8% for ring-stage parasites, imaging with near-UV light on a standard microscope resulted in 96.8% overall accuracy and over 90% recall for ring-stage parasites. Both imaging systems were tested extrinsically by parasitemia titration, revealing superior performance over manually-scored Giemsa-stained smears, and a limit of detection below 0.1%. Our results establish that label-free parasitemia analysis of live cells is possible in a biomedical laboratory setting without the need for complex optical instrumentation. We anticipate future extensions of this work could enable label-free clinical diagnostic measurements, one day eliminating the need for conventional blood smear analysis.

Hemoglobin quantification in red blood cells via dry mass mapping based on UV absorption.

SIGNIFICANCE: The morphological properties and hemoglobin (Hb) content of red blood cells (RBCs) are essential biomarkers to diagnose or monitor various types of hematological disorders. Label-free mass mapping approaches enable accurate Hb quantification from individual cells, serving as promising alternatives to conventional hematology analyzers. Deep ultraviolet (UV) microscopy is one such technique that allows high-resolution, molecular imaging, and absorption-based mass mapping. AIM: To compare UV absorption-based mass mapping at four UV wavelengths and understand variations across wavelengths and any assumptions necessary for accurate Hb quantification. APPROACH: Whole blood smears are imaged with a multispectral UV microscopy system, and the RBCs' dry masses are computed. This approach is compared to quantitative phase imaging-based mass mapping using data from an interferometric UV imaging system. RESULTS: Consistent Hb mass and mean corpuscular Hb values are obtained at all wavelengths, with the precision of the single-cell mass measurements being nearly identical at 220, 260, and 280 nm but slightly lower at 300 nm. CONCLUSIONS: A full hematological analysis (including white blood cell identification and characterization, and Hb quantification) may be achieved using a single UV illumination wavelength, thereby improving the speed and cost-effectiveness.

Initial Leuprolide Acetate Release from Poly(d,l-lactide-co-glycolide) in Situ Forming Implants as Studied by Ultraviolet-Visible Imaging.

The initial drug release from in situ forming implants is affected by factors such as the physicochemical properties of the active pharmaceutical ingredient, the type of the excipients utilized, and the surrounding environment. The feasibility of UV-vis imaging for characterization of the initial behavior of poly(d,l-lactide-co-glycolide) (PLGA)/1-methyl-2-pyrrolidinone (NMP) in situ forming implants was investigated. The in vitro release of leuprolide acetate (LA) and implant formation in real time were monitored using dual-wavelength imaging at 280 and 525 nm, respectively, in matrices based on agarose gel and hyaluronic acid (HA) solution emulating the subcutaneous matrix. Three hours upon injection of the pre-formulation, approximately 15% of the total amount of LA administered was found in the agarose gel, while 5% was released from the implant into the HA solution. Concurrently, more extensive swelling of the implants in the HA solution as compared to implants in the agarose gel was observed. Transport of both LA and the solvent NMP was investigated using UV-vis imaging in a small-scale cell where the geometry of the formulation was controlled, showing a linear correlation between drug release and solvent escape. Light microscopy showed that the microstructures of the resulting implants in agarose gel and HA solution were different, which may be attributed to the different solvent exchange rates. UV imaging was also used to examine the interaction of LA with the release medium by characterizing the diffusion of LA in agarose gel, HA solution, and phosphate buffered saline. The reduced LA diffusivity in HA solution as compared to agarose gel and the LA distribution coefficient in the agarose gel-HA system indicated the presence of interactions between LA and HA. Our findings show that the external environment affects the solvent exchange kinetics for in situ forming implants in vitro, resulting in different types of initial release behavior. UV-vis imaging in combination with biorelevant matrices may offer an interesting approach in the development of in situ forming implant delivery systems.

Label-free hematology analysis using deep-ultraviolet microscopy.

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.

TADs and Their Borders: Free Movement or Building a Wall?

The tridimensional (3D) organization of mammalian genomes combines structures from different length scales. Within this organization, Topologically Associating Domains (TADs) are visible in Hi-C heat maps at the sub-megabase scale. The integrity of TADs is important for correct gene expression, but in a context-dependent and variable manner. The correct structure and function of TADs require the binding of the CTCF protein at both borders, which appears to block an active and dynamic mechanism of "Cohesin-mediated loop extrusion." As a result, mammalian TADs appear as so-called "loop domains" in Hi-C data, which are the focus of this review. Here, we present a reanalysis of TADs from three "golden-standard" mammalian Hi-C data sets. Despite the prominent presence of TADs in Hi-C heat maps from all studies, we find consistently that regions within these domains are only moderately insulated from their surroundings. Moreover, single-cell Hi-C and superresolution microscopy have revealed that the structure of TADs and the position of their borders can vary from cell to cell. The function of TADs as units of gene regulation may thus require additional aspects, potentially incorporating the mechanism of loop extrusion as well. Recent developments in single-cell and multi-contact genomics and superresolution microscopy assays will be instrumental to link TAD formation and structure to their function in transcriptional regulation.

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine.

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

Three-Dimensional Microscopy by Milling with Ultraviolet Excitation.

Analysis of three-dimensional biological samples is critical to understanding tissue function and the mechanisms of disease. Many chronic conditions, like neurodegenerative diseases and cancers, correlate with complex tissue changes that are difficult to explore using two-dimensional histology. While three-dimensional techniques such as confocal and light-sheet microscopy are well-established, they are time consuming, require expensive instrumentation, and are limited to small tissue volumes. Three-dimensional microscopy is therefore impractical in clinical settings and often limited to core facilities at major research institutions. There would be a tremendous benefit to providing clinicians and researchers with the ability to routinely image large three-dimensional tissue volumes at cellular resolution. In this paper, we propose an imaging methodology that enables fast and inexpensive three-dimensional imaging that can be readily integrated into current histology pipelines. This method relies on block-face imaging of paraffin-embedded samples using deep-ultraviolet excitation. The imaged surface is then ablated to reveal the next tissue section for imaging. The final image stack is then aligned and reconstructed to provide tissue models that exceed the depth and resolution achievable with modern three-dimensional imaging systems.

Ex Vivo Microscopy: A Promising Next-Generation Digital Microscopy Tool for Surgical Pathology Practice.

CONTEXT.­: The rapid evolution of optical imaging modalities in recent years has opened the opportunity for ex vivo tissue imaging, which has significant implications for surgical pathology practice. These modalities have promising potential to be used as next-generation digital microscopy tools for examination of fresh tissue, with or without labeling with contrast agents. OBJECTIVE.­: To review the literature regarding various types of ex vivo optical imaging platforms that can generate digital images for tissue recognition with potential for utilization in anatomic pathology clinical practices. DATA SOURCES.­: Literature relevant to ex vivo tissue imaging obtained from the PubMed database. CONCLUSIONS.­: Ex vivo imaging of tissues can be performed by using various types of optical imaging techniques. These next-generation digital microscopy tools have a promising potential for utilization in surgical pathology practice.

Simple ultraviolet microscope using off-the-shelf components for point-of-care diagnostics.

At the primary care setting, where there are often no or minimal laboratories, examinations often consist of self-testing and rapid diagnostics. Because of this, medical devices must be simple, robust, and easy to operate. To address these concerns, an alternate fluorescence microscope design uses ultraviolet (UV) excitation, since fluorescent dyes that are excitable in the visible region are also excitable by UV. This may allow for the removal of typical excitation, emission, and dichroic filters as optical components absorb UV wavelengths and UV is not detected by silicon based detectors. Additionally, UV has a very low penetration into samples, which may allow for controlling the depth of excitation, and thus the imaging volume. Based on these ideas, we developed a simple fluorescence microscope built completely from off-the-shelf components that uses UV to image fluorescently stained samples. The simple opto-mechanical design of the system may allow it to be more compact and easy to use, as well as decrease the overall cost of the diagnostic device. For biological validation, we imaged whole blood stained with acridine orange and performed a two-part white blood cell differential count.

Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins.

Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.

Adaptation of microscopy with ultraviolet surface excitation for enhancing STEM and undergraduate education.

Microscopy with ultraviolet surface excitation (MUSE) is investigated as a means to enhance curricula and education in the life sciences based on simplicity of use, the incorporation of inexpensive hardware, and the simplest methods of tissue preparation. Ultraviolet excitation in effect replaces tissue sectioning because it penetrates only a few micrometers below the tissue surface at the single cell level, preventing the generation of out-of-focus light. Although tissue autofluorescence may be used, image quality and content can be enhanced by a brief immersion in a solution of nontoxic fluorescent dyes that selectively highlight different cellular compartments. Safe mixed-dye powder combinations have been developed to provide students who have minimal lab proficiencies with a one-step tissue staining process for rapid tissue preparation.

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.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation.

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Penetración Dentinaria in vitro del Hipoclorito de Sodio a Diferentes Concentraciones con las Técnicas de Irrigación Convencional y Ultrasónica Pasiva / In vitro Dentinary Penetration of Sodium Hypochlorite at Different Concentrations with the Conventional and Ultrasonic Passive Irrigation Techniques

RESUMEN: El objetivo del presente trabajo fue comparar la penetración dentinaria in vitro entre las concentraciones de hipoclorito de sodio (NaClO) al 5 % y al 2,5 % con técnicas de irrigación convencional e irrigación ultrasónica pasiva. Este fue un estudio transversal, prospectivo y experimental. Se trabajó con 40 segmentos radiculares (especímenes) de 5 mm de longitud, divididos en 4 grupos. Todos los especímenes se sumergieron en violeta cristal durante 24 horas. Se realizó la preparación biomecánica con sistema rotatorio Pro Taper. Al primer grupo se irrigó con NaClO al 2,5 % con irrigación convencional, al segundo grupo con NaClO al 2,5 % con irrigación convencional más irrigación ultrasónica pasiva por 30 segundos, al tercer grupo con NaClO al 5 % con irrigación convencional y al cuarto grupo con NaClO al 5 % con irrigación convencional más irrigación ultrasónica pasiva por 30 segundos. Al final de la preparación a todos los especímenes se les realizó un corte mesio distal. La profundidad de la penetración del NaClO fue deterrminado por el blanqueamiento de la mancha y medido con un microscopio de luz con una magnificación de 40 X. La prueba estadística usada fue ANOVA con un nivel de significancia de 5 %. El NaClO al 5 % con técnica de irrigación ultrasónica pasiva presentó la mayor penetración dentinaria in vitro.

ABSTRACT: The objective of the present study was to compare in vitro dentin penetration between 5 % and 2.5 % sodium hypochlorite (NaClO) concentrations using conventional irrigation and passive ultrasonic irrigation techniques. This was a cross-sectional, prospective and experimental study. It was worked with 40 root segments (specimen) of 5 mm length, divided into 4 groups. All specimens were immersed in crystal violet for 24 hours. The biomechanical preparation was performed with Pro Taper rotary system. The first group was irrigated with 2.5 % NaClO with conventional irrigation, the second group with 2.5 % NaClO with conventional irrigation plus ulrasonic passive irrigation for 30 seconds, the third group with 5 % NaClO with conventional irrigation and the fourth with 5 % NaClO with conventional irrigation plus ultrasonic passive irrigation for 30 seconds. At the end of the preparation, all specimens had a distal mesio cut. The depth of NaClO penetration was determined by bleaching of the stain and measured with a light microscope at a magnification of 40 X. The statistical test used was ANOVA with a significance level of 5 %. The 5 % NaClO with passive ultrasonic irrigation showed the highest dentin penetration in vitro.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.