A Review of Pathology and Analysis of Approaches to Easing Kidney Disease Impact: Host-Pathogen Communication and Biomedical Visualization Perspective : Advanced Microscopy and Visualization of Host-Pathogen Communication.

INTRODUCTION: In addition to affecting the upper respiratory tract, severe acute respiratory syndrome-coronavirus (SARS-CoV) and SARS-CoV-2) can target kidneys resulting in disease impact. There is a lack of effective treatment for SARs-CoV and SARS-CoV-2, and so one approach could be to consider to lower the probable risk and onset of disease amongst immunocompromised and immunosuppressed individuals and patients. Angiotensin Converting Enzyme 2 (ACE2) has a promising impact including acting against SARs-CoV and SARS-CoV-2 symptoms. Current literature states that ACE2 is expressed across several physiological systems, including the lungs, cardiovascular, gut, kidneys, and central nervous, and across endothelia. AIMS: This chapter seeks to investigate causes and potential mechanisms during SARS infection (CoV-2), renal interaction, and the effects of acute kidney Injury (AKI). OBJECTIVES: This chapter will provide an overview of microscopy and visualization of host-pathogen communication and principles of ACE2 in the context of immunology and impact on renal pathophysiology. DESIGN: This chapter focuses to provide basic principles of ACE2 and the analysis and effect of immunology and pathological components important in relation to SARs infection. DISCUSSION: There has been a surge in literature surrounding mechanisms attributing to SARS-CoV and SARS-CoV-2 action on immune response to pathogens. There is an advantage to implementing ACE2 treatment to improve immune response against infection. CONCLUSION: ACE2 may provide appropriate strategies for the management of symptoms that relate to SARS-CoV and SARS-CoV-2 in most immunocompromised or immunosuppressed patients. Visualization of ACE2 action can be achieved through microscopy to understand host-pathogen communication.

Multi-Illumination Single-Holographic-Exposure Lensless Fresnel (MISHELF) Microscopy: Principles and Biomedical Applications.

Lensless holographic microscopy (LHM) comes out as a promising label-free technique since it supplies high-quality imaging and adaptive magnification in a lens-free, compact and cost-effective way. Compact sizes and reduced prices of LHMs make them a perfect instrument for point-of-care diagnosis and increase their usability in limited-resource laboratories, remote areas, and poor countries. LHM can provide excellent intensity and phase imaging when the twin image is removed. In that sense, multi-illumination single-holographic-exposure lensless Fresnel (MISHELF) microscopy appears as a single-shot and phase-retrieved imaging technique employing multiple illumination/detection channels and a fast-iterative phase-retrieval algorithm. In this contribution, we review MISHELF microscopy through the description of the principles, the analysis of the performance, the presentation of the microscope prototypes and the inclusion of the main biomedical applications reported so far.

SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells.

It is urgent to understand the infection mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for the prevention and treatment of COVID-19. The infection of SARS-CoV-2 starts when the receptor-binding domain (RBD) of viral spike protein binds to angiotensin-converting enzyme 2 (ACE2) of the host cell, but the endocytosis details after this binding are not clear. Here, RBD and ACE2 were genetically coded and labeled with organic dyes to track RBD endocytosis in living cells. The photostable dyes enable long-term structured illumination microscopy (SIM) imaging and to quantify RBD-ACE2 binding (RAB) by the intensity ratio of RBD/ACE2 fluorescence. We resolved RAB endocytosis in living cells, including RBD-ACE2 recognition, cofactor-regulated membrane internalization, RAB-bearing vesicle formation and transport, RAB degradation, and downregulation of ACE2. The RAB was found to activate the RBD internalization. After vesicles were transported and matured within cells, RAB was finally degraded after being taken up by lysosomes. This strategy is a promising tool to understand the infection mechanism of SARS-CoV-2.

A photonic resonator interferometric scattering microscope for label-free detection of nanometer-scale objects with digital precision in point-of-use environments.

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic source. The use of a photonic crystal substrate for interferemetric scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instrument's main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position. In this work, we characterize the system's performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosome, and ferritin protein.

COVID-19 Adaptations with Virtual Microscopy.

This chapter aims to discuss and compare the different approaches used to teach histology to dental students before and during the COVID-19 pandemic and reflect on the best practices to be retained. Prior to the COVID-19 pandemic, the University of Glasgow School of Dentistry converted its large and unique collections of microscopy slides into digital files to curate this unique asset and protect it for prosperity. Initially, a virtual microscopy (VM) educational platform was purchased to allow digital teaching of histology, oral biology, and oral pathology. Prior to COVID-19, dental undergraduate students received VM teaching via a blended learning approach with theoretical content preceding a practical discussion session using VM. Some teachers in later years of the dental course experimented with flipped class strategies. At the beginning of 2020, with the lockdown restrictions imposed, the teaching content all had to move to remote online learning with virtual sessions, recorded video classes, online content, videotelephony, and online chat, allowing the students to undertake the content asynchronously and remotely. To overcome the interactive limitations of online delivery, a Microsoft Team was created in some sessions and used to support active small group learning and teaching of general histology allowing students to share histological annotations with their peers and tutors. The experience of teaching histology using only virtual and online content has had a positive academic outcome for the students as all first year students had passed their exams. However, we also recognise several limitations, such as the restrictive interpersonal interaction using videotelephony and online chat as well as the ad hoc feedback. The processes used and the challenges and benefits of VM will be discussed in this chapter.

Malaria diagnostic methods with the elimination goal in view.

Malaria control measures have been in use for years but have not completely curbed the spread of infection. Ultimately, global elimination is the goal. A major playmaker in the various approaches to reaching the goal is the issue of proper diagnosis. Various diagnostic techniques were adopted in different regions and geographical locations over the decades, and these have invariably produced diverse outcomes. In this review, we looked at the various approaches used in malaria diagnostics with a focus on methods favorably used during pre-elimination and elimination phases as well as in endemic regions. Microscopy, rapid diagnostic testing (RDT), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR) are common methods applied depending on prevailing factors, each with its strengths and limitations. As the drive toward the elimination goal intensifies, the search for ideal, simple, fast, and reliable point-of-care diagnostic tools is needed more than ever before to be used in conjunction with a functional surveillance system supported by the ideal vaccine.

Extracting multiple surfaces from 3D microscopy images in complex biological tissues with the Zellige software tool.

BACKGROUND: Efficient tools allowing the extraction of 2D surfaces from 3D-microscopy data are essential for studies aiming to decipher the complex cellular choreography through which epithelium morphogenesis takes place during development. Most existing methods allow for the extraction of a single and smooth manifold of sufficiently high signal intensity and contrast, and usually fail when the surface of interest has a rough topography or when its localization is hampered by other surrounding structures of higher contrast. Multiple surface segmentation entails laborious manual annotations of the various surfaces separately. RESULTS: As automating this task is critical in studies involving tissue-tissue or tissue-matrix interaction, we developed the Zellige software, which allows the extraction of a non-prescribed number of surfaces of varying inclination, contrast, and texture from a 3D image. The tool requires the adjustment of a small set of control parameters, for which we provide an intuitive interface implemented as a Fiji plugin. CONCLUSIONS: As a proof of principle of the versatility of Zellige, we demonstrate its performance and robustness on synthetic images and on four different types of biological samples, covering a wide range of biological contexts.

An intra-cytoplasmic route for SARS-CoV-2 transmission unveiled by Helium-ion microscopy.

SARS-CoV-2 virions enter the host cells by docking their spike glycoproteins to the membrane-bound Angiotensin Converting Enzyme 2. After intracellular assembly, the newly formed virions are released from the infected cells to propagate the infection, using the extra-cytoplasmic ACE2 docking mechanism. However, the molecular events underpinning SARS-CoV-2 transmission between host cells are not fully understood. Here, we report the findings of a scanning Helium-ion microscopy study performed on Vero E6 cells infected with mNeonGreen-expressing SARS-CoV-2. Our data reveal, with unprecedented resolution, the presence of: (1) long tunneling nanotubes that connect two or more host cells over submillimeter distances; (2) large scale multiple cell fusion events (syncytia); and (3) abundant extracellular vesicles of various sizes. Taken together, these ultrastructural features describe a novel intra-cytoplasmic connection among SARS-CoV-2 infected cells that may act as an alternative route of viral transmission, disengaged from the well-known extra-cytoplasmic ACE2 docking mechanism. Such route may explain the elusiveness of SARS-CoV-2 to survive from the immune surveillance of the infected host.

Novel TB smear microscopy automation system in detecting acid-fast bacilli for tuberculosis - A multi-center double blind study.

Due to COVID-19 pandemic, there is a large global drop in the number of newly diagnosed cases with tuberculosis (TB) worldwide. Actions to mitigate and reverse the impact of the COVID-19 pandemic on TB are urgently needed. Recent development of TB smear microscopy automation systems using artificial intelligence may increase the sensitivity of TB smear microscopy. The objective is to evaluate the performance of an automation system (µ-Scan 2.0, Wellgen Medical) over manual smear microscopy in a multi-center, double-blind trial. Total of 1726 smears were enrolled. Referee medical technician and culture served as primary and secondary gold standards for result discrepancy. Results showed that, compared to manual microscopy, the µ-Scan 2.0's performance of accuracy, sensitivity and specificity were 95.7% (1651/1726), 87.7% (57/65), and 96.0% (1594/1661), respectively. The negative predictive value was 97.8% at prevalence of 8.2%. Manual smear microscopy remains the primary diagnosis of pulmonary tuberculosis (TB). Use of automation system could achieve higher TB smear sensitivity and laboratory efficiency. It can also serve as a screening tool that complements molecular methods to reduce the total cost for TB diagnosis and control. Furthermore, such automation system is capable of remote access by internet connection and can be deployed in area with limited medical resources.

Gires-Tournois Immunoassay Platform for Label-Free Bright-Field Imaging and Facile Quantification of Bioparticles.

Bright-field imaging of nanoscale bioparticles is a challenging task for optical microscopy because the light-matter interactions of bioparticles are weak on conventional surfaces due to their low refractive index and small size. Alternatively, advanced imaging techniques, including near-field microscopy and phase microscopy, have enabled visualization and quantification of the bioparticles, but they require assistance of sophisticated/customized systems and post-processing with complex established algorithms. Here, a simple and fast immunoassay device, Gires-Tournois immunoassay platform (GTIP) is presented, which provides unique color dynamics in response to optical environment changes and thus enables the label-free bright-field imaging and facile quantification of bioparticles using conventional optical microscopy. Bioparticles on GTIP slow down the velocity of reflected light, leading to vivid color change according to the local particle density and maximizing chromatic contrast for high spatial distinguishability. The particle distribution and density on the surface of the resonator are readily analyzed through 2D raster-scanning-based chromaticity analysis. GTIP offers multiscale sensing capability for target analytes that possess different refractive indices and sizes.

CapillaryNet: An automated system to quantify skin capillary density and red blood cell velocity from handheld vital microscopy.

Capillaries are the smallest vessels in the body which are responsible for delivering oxygen and nutrients to surrounding cells. Various life-threatening diseases are known to alter the density of healthy capillaries and the flow velocity of erythrocytes within the capillaries. In previous studies, capillary density and flow velocity were manually assessed by trained specialists. However, manual analysis of a standard 20-s microvascular video requires 20 min on average and necessitates extensive training. Thus, manual analysis has been reported to hinder the application of microvascular microscopy in a clinical environment. To address this problem, this paper presents a fully automated state-of-the-art system to quantify skin nutritive capillary density and red blood cell velocity captured by handheld-based microscopy videos. The proposed method combines the speed of traditional computer vision algorithms with the accuracy of convolutional neural networks to enable clinical capillary analysis. The results show that the proposed system fully automates capillary detection with an accuracy exceeding that of trained analysts and measures several novel microvascular parameters that had eluded quantification thus far, namely, capillary hematocrit and intracapillary flow velocity heterogeneity. The proposed end-to-end system, named CapillaryNet, can detect capillaries at ~0.9 s per frame with ~93% accuracy. The system is currently used as a clinical research product in a larger e-health application to analyse capillary data captured from patients suffering from COVID-19, pancreatitis, and acute heart diseases. CapillaryNet narrows the gap between the analysis of microcirculation images in a clinical environment and state-of-the-art systems.

OME-NGFF: a next-generation file format for expanding bioimaging data-access strategies.

The rapid pace of innovation in biological imaging and the diversity of its applications have prevented the establishment of a community-agreed standardized data format. We propose that complementing established open formats such as OME-TIFF and HDF5 with a next-generation file format such as Zarr will satisfy the majority of use cases in bioimaging. Critically, a common metadata format used in all these vessels can deliver truly findable, accessible, interoperable and reusable bioimaging data.

Label-Free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 103 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Digital Pathology During the COVID-19 Outbreak in Italy: Survey Study.

BACKGROUND: Transition to digital pathology usually takes months or years to be completed. We were familiarizing ourselves with digital pathology solutions at the time when the COVID-19 outbreak forced us to embark on an abrupt transition to digital pathology. OBJECTIVE: The aim of this study was to quantitatively describe how the abrupt transition to digital pathology might affect the quality of diagnoses, model possible causes by probabilistic modeling, and qualitatively gauge the perception of this abrupt transition. METHODS: A total of 17 pathologists and residents participated in this study; these participants reviewed 25 additional test cases from the archives and completed a final psychologic survey. For each case, participants performed several different diagnostic tasks, and their results were recorded and compared with the original diagnoses performed using the gold standard method (ie, conventional microscopy). We performed Bayesian data analysis with probabilistic modeling. RESULTS: The overall analysis, comprising 1345 different items, resulted in a 9% (117/1345) error rate in using digital slides. The task of differentiating a neoplastic process from a nonneoplastic one accounted for an error rate of 10.7% (42/392), whereas the distinction of a malignant process from a benign one accounted for an error rate of 4.2% (11/258). Apart from residents, senior pathologists generated most discrepancies (7.9%, 13/164). Our model showed that these differences among career levels persisted even after adjusting for other factors. CONCLUSIONS: Our findings are in line with previous findings, emphasizing that the duration of transition (ie, lengthy or abrupt) might not influence the diagnostic performance. Moreover, our findings highlight that senior pathologists may be limited by a digital gap, which may negatively affect their performance with digital pathology. These results can guide the process of digital transition in the field of pathology.