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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 -
Microbiology Spectrum May 2019The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and... (Review)
Review
The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.
Topics: Bacterial Capsules; Bacteriological Techniques; Cell Membrane; Cell Wall; Cryoelectron Microscopy; Electron Microscope Tomography; Extracellular Vesicles; Freezing; Gram-Positive Bacteria; Imaging, Three-Dimensional; Lipopolysaccharides; Microscopy, Electron; Microscopy, Electron, Transmission; Mycobacterium; Peptidoglycan; Teichoic Acids
PubMed: 31124431
DOI: 10.1128/microbiolspec.GPP3-0044-2018 -
Quarterly Reviews of Biophysics May 1988
Review
Topics: Animals; Freezing; Histological Techniques; Humans; Microscopy, Electron; Tissue Preservation
PubMed: 3043536
DOI: 10.1017/s0033583500004297 -
Nature Methods Jan 2019Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an...
Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
Topics: Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Stereoisomerism
PubMed: 30559430
DOI: 10.1038/s41592-018-0238-1 -
Acta Crystallographica. Section D,... Feb 2018Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of... (Review)
Review
Macromolecular crystallography (MX) has been a motor for biology for over half a century and this continues apace. A series of revolutions, including the production of recombinant proteins and cryo-crystallography, have meant that MX has repeatedly reinvented itself to dramatically increase its reach. Over the last 30 years synchrotron radiation has nucleated a succession of advances, ranging from detectors to optics and automation. These advances, in turn, open up opportunities. For instance, a further order of magnitude could perhaps be gained in signal to noise for general synchrotron experiments. In addition, X-ray free-electron lasers offer to capture fragments of reciprocal space without radiation damage, and open up the subpicosecond regime of protein dynamics and activity. But electrons have recently stolen the limelight: so is X-ray crystallography in rude health, or will imaging methods, especially single-particle electron microscopy, render it obsolete for the most interesting biology, whilst electron diffraction enables structure determination from even the smallest crystals? We will lay out some information to help you decide.
Topics: Crystallography; Macromolecular Substances; Microscopy, Electron; Microscopy, Electron, Transmission; Synchrotrons
PubMed: 29533241
DOI: 10.1107/S2059798317016709 -
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 -
Current Protocols in Cytometry Oct 2014Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with... (Comparative Study)
Comparative Study Review
Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation--integration.
Topics: Animals; Humans; Microscopy, Electron; Microscopy, Fluorescence
PubMed: 25271959
DOI: 10.1002/0471142956.cy1236s70 -
Platelets Jul 2020Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and... (Review)
Review
Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and scanning electron microscopy (EM) provided a foundation for our initial understanding of platelet structure and how it changes upon platelet activation. EM approaches have since been utilized to study platelets and thrombi in the context of basic, translational and clinical research, and they are instrumental in the diagnosis of multiple platelet function disorders. In this brief review, we provide a sampling of the many contributions EM based studies have made to the field, including both historical highlights and contemporary applications. We will also discuss exciting new imaging modalities based on EM and their utility for the study of platelets, hemostasis and thrombosis into the future.
Topics: Blood Platelets; Hemostasis; Humans; Microscopy, Electron; Thrombosis
PubMed: 32423268
DOI: 10.1080/09537104.2020.1763939 -
Annual Review of Physical Chemistry Apr 2021We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and... (Review)
Review
We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.
Topics: Caulobacter crescentus; Cryoelectron Microscopy; Electron Microscope Tomography; HEK293 Cells; Humans; Microscopy, Electron; Microscopy, Fluorescence; Nanotechnology; Single Molecule Imaging; Subcellular Fractions
PubMed: 33441030
DOI: 10.1146/annurev-physchem-090319-051546 -
ELife Aug 2022A combination of light and electron microscopy has revealed further details about the location and interactions of hematopoietic stem and progenitor cells.
A combination of light and electron microscopy has revealed further details about the location and interactions of hematopoietic stem and progenitor cells.
Topics: Hematopoietic Stem Cells; Microscopy, Electron; Stem Cell Niche
PubMed: 35983923
DOI: 10.7554/eLife.81963