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The Journal of Investigative Dermatology Feb 2022Volume scanning electron microscopy (VSEM) involves the serial sectioning and imaging of a sample using scanning electron microscopy (SEM), followed by segmentation and... (Review)
Review
Volume scanning electron microscopy (VSEM) involves the serial sectioning and imaging of a sample using scanning electron microscopy (SEM), followed by segmentation and three-dimensional (3D) reconstruction using computer software packages to allow visualization of 3D structures. VSEM can reveal qualitative and quantitative properties of organelles and cells within tissues at nanoscale. The ability to visualize spatial relationships of structures of interest within and across cells in 3D space in particular sets VSEM apart from conventional SEM and transmission electron microscopy. Here, we provide an overview of VSEM platforms and image processing, highlighting characteristics that will aid selection of a method to address specific research questions in dermatological research.
Topics: Animals; Biomedical Research; Dermatology; HEK293 Cells; Humans; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Skin
PubMed: 34762923
DOI: 10.1016/j.jid.2021.10.020 -
PloS One 2013Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized...
Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.
Topics: Cell Line, Tumor; Humans; Microscopy, Electron, Scanning; Microscopy, Fluorescence
PubMed: 23409024
DOI: 10.1371/journal.pone.0055707 -
Frontiers in Neural Circuits 2018One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron... (Review)
Review
One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.
Topics: Animals; Brain; Humans; Image Processing, Computer-Assisted; Microscopy, Electron, Scanning; Microtomy; Nanotubes; Nerve Net
PubMed: 30483066
DOI: 10.3389/fncir.2018.00098 -
PloS One 2013The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol...
The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.
Topics: Actins; Animals; Cryoultramicrotomy; Imaging, Three-Dimensional; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microtomy; Mitochondria; NIH 3T3 Cells; Nuclear Lamina; Peroxisomes; Phalloidine; Staining and Labeling
PubMed: 24204771
DOI: 10.1371/journal.pone.0077209 -
Frontiers in Neural Circuits 2019Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells....
Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.
Topics: Animals; Brain; Callithrix; Mice, Inbred C57BL; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Neuroimaging
PubMed: 31133819
DOI: 10.3389/fncir.2019.00029 -
Scientific Reports Feb 2020Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread...
Phenotypic heterogeneity is an important trait for the development and survival of many microorganisms including the yeast Cryptococcus spp., a deadly pathogen spread worldwide. Here, we have applied scanning electron microscopy (SEM) to define four Cryptococcus spp. capsule morphotypes, namely Regular, Spiky, Bald, and Phantom. These morphotypes were persistently observed in varying proportions among yeast isolates. To assess the distribution of such morphotypes we implemented an automated pipeline capable of (1) identifying potentially cell-associated objects in the SEM-derived images; (2) computing object-level features; and (3) classifying these objects into their corresponding classes. The machine learning approach used a Random Forest (RF) classifier whose overall accuracy reached 85% on the test dataset, with per-class specificity above 90%, and sensitivity between 66 and 94%. Additionally, the RF model indicates that structural and texture features, e.g., object area, eccentricity, and contrast, are most relevant for classification. The RF results agree with the observed variation in these features, consistently also with visual inspection of SEM images. Finally, our work introduces morphological variants of Cryptococcus spp. capsule. These can be promptly identified and characterized using computational models so that future work may unveil morphological associations with yeast virulence.
Topics: Anatomic Variation; Cryptococcus; Fungal Capsules; Machine Learning; Microscopy, Electron, Scanning; Phenotype
PubMed: 32047210
DOI: 10.1038/s41598-020-59276-w -
Advanced Biology Aug 2023Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique... (Review)
Review
Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.
Topics: Microscopy, Electron, Scanning; Humans; Animals; Artificial Intelligence
PubMed: 37246236
DOI: 10.1002/adbi.202300139 -
Journal of Microbiology, Immunology,... Oct 2022To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct...
To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.
Topics: Humans; Microscopy, Electron, Scanning; Ascomycota; Biofilms; Plant Extracts
PubMed: 34836818
DOI: 10.1016/j.jmii.2021.11.002 -
Journal of Dairy Science Dec 2020The surface epithelium of the bovine endometrium comprises at least 2 cell types (ciliated cells and secretory cells with microvilli), but their distribution and...
The surface epithelium of the bovine endometrium comprises at least 2 cell types (ciliated cells and secretory cells with microvilli), but their distribution and morphological changes over the estrous cycle are poorly understood. The objective was to quantify the number of ciliated cells and assess morphological changes in secretory cells on the uterine surface epithelium during the estrous cycle. Caruncular endometrium (CAR) and intercaruncular endometrium (ICAR) samples were collected from the uterine body, the horn ipsilateral to the corpus luteum or dominant follicle (H-CL/DF), and the horn contralateral to the corpus luteum or dominant follicle (H-NCL/NDF) from heifers following slaughter on d 0 (estrus; n = 5) or d 14 (mid-luteal phase; n = 5) of the estrous cycle. Samples were prepared for scanning electron microscopy at 1,000× magnification. Four to 10 fields (256 × 225 µm) for each sample were examined (n = 567 images). The number of ciliated cells was counted and the surface was scored for the morphology of the secretory cells (0 = absence of microvilli on surface; 3 = 100% of surface covered with microvilli). Ciliated cells were present in both the CAR and ICAR regions. The number of ciliated cells per field increased from d 0 to 14 in CAR and decreased from d 0 to14 in ICAR. The scanning electron microscopy revealed a general lack of uniformity in the lawn of microvilli on the surface of the endometrium. Based on the scores, approximately 25% of the fields had a surface that was <50% covered by microvilli. Depletion of microvilli may be explained by a normal process where apical protrusions are formed and either regress back into the cell surface or break to release their contents into the uterine lumen. These studies support the hypothesis that the surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface. The morphology of the apical surface may have a key role in governing pregnancy establishment.
Topics: Animals; Cattle; Corpus Luteum; Endometrium; Epithelium; Estrous Cycle; Female; Luteal Phase; Microscopy, Electron, Scanning; Microvilli; Pregnancy
PubMed: 32981737
DOI: 10.3168/jds.2020-18852 -
Turkiye Parazitolojii Dergisi Sep 2018The aim of this study was to examine the morphological characteristics of Colpocephalum nanum (C. nanum) Piaget, 1890 using light microscopy (LM) and scanning electron...
OBJECTIVE
The aim of this study was to examine the morphological characteristics of Colpocephalum nanum (C. nanum) Piaget, 1890 using light microscopy (LM) and scanning electron microscopy (SEM).
METHODS
For this purpose, the C. nanum specimens collected from long-legged buzzards, Buteo rufinus (B. rufinus) (Accipitriformes: Accipitridae), in Turkey were examined under LM and SM for morphological characteristics. The specimens were fixed and kept in 70% ethanol, cleared and mounted on the slides in Canada balsam. They were examined for morphological characteristics under LM. Some of the samples were put in a plate on absorbing paper and kept overnight, for ether to evaporate. These samples were mounted on aluminum stubs to study the ventral surface by placing them on their dorsal or ventral surface on double-sided adhesive tape. They were sputter-coated three times with gold, each time for approximately 7 minutes and later viewed using SEM (Zeiss SUPRA 55 VP FE-SEM and Zeiss EVO lS 10).
RESULTS
Parts of the specimens were photographed, and the obtained data about morphological characteristic were evaluated in detail.
CONCLUSION
The LM and SEM photos of C. nanum were compared, and information about the important criteria for diagnosis and other morphological characteristics was obtained.
Topics: Amblycera; Animals; Bird Diseases; Falconiformes; Lice Infestations; Microscopy; Microscopy, Electron, Scanning; Turkey
PubMed: 30280693
DOI: 10.5152/tpd.2018.5808