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Current Protocols in Microbiology May 2012Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful...
Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This unit describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM.
Topics: Biomedical Research; Image Processing, Computer-Assisted; Microscopy, Electron, Scanning; Specimen Handling
PubMed: 22549162
DOI: 10.1002/9780471729259.mc02b02s25 -
Folia Biologica 2021Prematurely erupted teeth are rare in fullterm neonates and extremely rare in prematurely delivered infants. The aim of this study was to present macroscopic and...
Prematurely erupted teeth are rare in fullterm neonates and extremely rare in prematurely delivered infants. The aim of this study was to present macroscopic and scanning electron microscopy (SEM) investigations of prematurely erupted primary teeth of preterm very low birthweight (VLBW) and extremely low birthweight (ELBW) infants. Three preterm VLBW and ELBW infants with prematurely erupted lower incisors were examined. The dental examination assessed the type, location, clinical appearance, and degree of mobility of the prematurely erupted teeth. The structural appearance of enamel and dentin of three extracted and longitudinally sectioned prematurely erupted teeth was investigated with scanning electron microscopy (SEM). Lower incisors were rootless with hypermobility. The surface of enamel of the prematurely erupted primary teeth was hypoplastic and hypomineralized and had a typical "honeycomb" appearance in SEM. The aprismatic type of enamel was visible in some regions. The neonatal line separating the layer of prenatal enamel from postnatal enamel was observed. The enamel prisms were interconnected by interprismatic substances, and cross-striations of prisms were visible. Dentin presented a typical tubular character. The dentinal layer near the enamel dentin junction had Y-shaped branching of dentinal tubules. On the pulpal side, dentin had a globular character. The macroscopic and SEM investigations particularly revealed alterations in enamel, while the dentin of neonatal teeth had a nearly normal appearance.
Topics: Dentin; Humans; Incisor; Infant; Infant, Newborn; Infant, Premature; Microscopy, Electron, Scanning
PubMed: 35151238
DOI: No ID Found -
STAR Protocols Sep 2021Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy...
Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy (SEM) of murine skin. The protocol enables characterization of the ultrastructure of the epidermis, dermis, hair follicles, basement membrane, and cell-cell junctions. We detail the specific steps for sample preparation and highlight the critical need for proper orientation of the sample for ultrathin sectioning. We also describe the isolation and preparation of primary keratinocyte monolayers for SEM. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2021).
Topics: Animals; Basement Membrane; Dermis; Epidermal Cells; Epidermis; Hair Follicle; Keratinocytes; Mice; Microscopy, Electron, Scanning; Skin
PubMed: 34458866
DOI: 10.1016/j.xpro.2021.100729 -
STAR Protocols Sep 2021This protocol enables correlative light and electron microscopy (CLEM) imaging of cell surface features without using dedicated equipment. Cells are cultured and fixed...
This protocol enables correlative light and electron microscopy (CLEM) imaging of cell surface features without using dedicated equipment. Cells are cultured and fixed on transparent substrates for confocal microscopy imaging. No conductive coating is employed in the scanning electron microscopy workflow, providing a clean cell surface observation, with fiducial markers assisting alignment of optical and topographical images. This protocol describes CLEM imaging for midbody remnants in MDCK cells but can also be applied to different cell types and surface features. For complete details on the use and execution of this protocol, please refer to Casares-Arias et al. (2020).
Topics: Animals; Cell Line; Cells, Cultured; Dogs; Electron Microscope Tomography; Imaging, Three-Dimensional; Madin Darby Canine Kidney Cells; Microscopy, Confocal; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Software; Workflow
PubMed: 34409307
DOI: 10.1016/j.xpro.2021.100727 -
BMC Biology Sep 2018Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in...
Array tomography encompasses light and electron microscopy modalities that offer unparalleled opportunities to explore three-dimensional cellular architectures in extremely fine structural and molecular detail. Fluorescence array tomography achieves much higher resolution and molecular multiplexing than most other fluorescence microscopy methods, while electron array tomography can capture three-dimensional ultrastructure much more easily and rapidly than traditional serial-section electron microscopy methods. A correlative fluorescence/electron microscopy mode of array tomography furthermore offers a unique capacity to merge the molecular discrimination strengths of multichannel fluorescence microscopy with the ultrastructural imaging strengths of electron microscopy. This essay samples the first decade of array tomography, highlighting applications in neuroscience.
Topics: Cytological Techniques; Electron Microscope Tomography; Imaging, Three-Dimensional; Microscopy, Electron, Scanning; Microscopy, Fluorescence
PubMed: 30189863
DOI: 10.1186/s12915-018-0560-1 -
Proceedings of the National Academy of... May 2023Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for...
Human islet primary cilia are vital glucose-regulating organelles whose structure remains uncharacterized. Scanning electron microscopy (SEM) is a useful technique for studying the surface morphology of membrane projections like cilia, but conventional sample preparation does not reveal the submembrane axonemal structure, which holds key implications for ciliary function. To overcome this challenge, we combined SEM with membrane-extraction techniques to examine primary cilia in native human islets. Our data show well-preserved cilia subdomains which demonstrate both expected and unexpected ultrastructural motifs. Morphometric features were quantified when possible, including axonemal length and diameter, microtubule conformations, and chirality. We further describe a ciliary ring, a structure that may be a specialization in human islets. Key findings are correlated with fluorescence microscopy and interpreted in the context of cilia function as a cellular sensor and communications locus in pancreatic islets.
Topics: Humans; Microscopy, Electron, Scanning; Cilia; Microscopy, Fluorescence; Islets of Langerhans; Microtubules
PubMed: 37205712
DOI: 10.1073/pnas.2302624120 -
Anais Brasileiros de Dermatologia 2021Cutis rhomboidalis nuchae was assessed in a 65-year-old patient. Optical microscopy showed basophilic agglomerations in the reticular dermis with decreased elastic...
Cutis rhomboidalis nuchae was assessed in a 65-year-old patient. Optical microscopy showed basophilic agglomerations in the reticular dermis with decreased elastic fibers. Transmission electron microscopy showed elongated, curved and fragmented structures, and in their interior the presence of electron-dense lumps was reduced and irregular, similar to modified elastic fibers, whereas the collagen fibers had a normal aspect. Scanning electron microscopy showed deposits between the bundles of collagen, resembling pebbles or stones. These findings demonstrate that, at one stage of the disease, the collagen remains normal and the alterations are seen in the elastic tissue.
Topics: Aged; Dermis; Elastic Tissue; Humans; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Skin Diseases
PubMed: 33775482
DOI: 10.1016/j.abd.2020.08.013 -
Poultry Science Jul 2021Poultry house dust is composed of fine particles which likely originate from a diverse range of materials such as feed, litter, excreta, and feathers. Little is known...
Poultry house dust is composed of fine particles which likely originate from a diverse range of materials such as feed, litter, excreta, and feathers. Little is known about the contribution of these sources to broiler house airborne dust so the present study was designed to identify the relative contributions of these sources. Samples of feed, excreta, feather, and bedding, known mixtures of these and settled dust from 28 broiler chicken flocks were tested for the concentration of 18 chemical elements. A chemometrics approach (the application of multivariate statistical techniques to chemical analysis data) was used to identify the primary source material in broiler chicken house dust samples. Scanning electron microscopy (SEM) was also used to analyze dust sample particulates based on examination of source materials. Excreta was found to be the main component of broiler chicken house dust, both by SEM and chemometric analysis. SEM of experimental flock dust between 7 and 35 days of age (d) revealed that the contribution of excreta to dust increased with age from 60% at 7 d to 95% at 28 d (P < 0.001). The proportion of bedding and feed in dust declined with age while the contribution of feather material remained low throughout. This study demonstrates that excreta provides the bulk of the material in poultry dust samples with bedding material, feed and feather material providing lower proportions. The relative contributions of these materials to dust varies with age of birds at dust collection. Additional research is required to determine the health and diagnostic implications of this variation.
Topics: Animals; Chickens; Dust; Feathers; Microscopy, Electron, Scanning; Poultry
PubMed: 34089932
DOI: 10.1016/j.psj.2021.101188 -
Scientific Reports Jun 2022The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for...
The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.
Topics: Argon; Histological Techniques; Microscopy, Electron, Scanning; Wood; Xylem
PubMed: 35650388
DOI: 10.1038/s41598-022-13122-3 -
Microscopy (Oxford, England) Oct 2022It is difficult to use scanning electron microscopy to observe the structure and movement of biological tissue immersed in the solution. To enable such observations, we...
It is difficult to use scanning electron microscopy to observe the structure and movement of biological tissue immersed in the solution. To enable such observations, we created a highly deformable and electron-transmissive polyimide film that can withstand the pressure difference between the high-vacuum electron column and the atmospheric-pressure sample chamber. With this film, we used scanning electron microscopy to measure the intrinsic fine structure and movement of the contractile fibers of excised mouse heart immersed in physiological solutions. Our measurements revealed that the excised heart is a dynamic tissue that undergoes relaxation oscillation based on a three-dimensional force balance.
Topics: Animals; Electrons; Mice; Microscopy, Electron, Scanning; Vacuum
PubMed: 35711152
DOI: 10.1093/jmicro/dfac030