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Correlative Light Electron Microscopy
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Customer Reviews. In either case, the target antigen may be a native protein or one that has been tagged with a marker peptide sequence.
Green fluorescent protein is a particularly useful epitope tag, as its fluorescence can be exploited to follow the localization of the marked protein in living cells. When necessary, the correlation can be made between fluorescence and electron micrographs of the same cell, before and after specimen preparation, a technique referred to as CLEM Nixon et al. Finally, reporting of immuno-EM staining results should be quantified by compiling data from a number of stained structures in various cells and samples, followed by checking for background signal with secondary antibody—only controls.
A fine example of this approach can be found in Rout et al. The stained sections on grids are now ready for examination in the electron microscope. A grid is inserted into the column of the electron microscope using a holder that places the grid in the electron beam.
Correlative Light and Electron Microscopy III
Modern electron microscopes have extensive, user-friendly computer interfaces, making it relatively simple to learn how to safely operate the instruments. Digital cameras not only record the images but are also used for locating areas of interest, focusing, and correcting astigmatism and have almost universally replaced traditional film. Newer EMs have software tools that allow the user to map the grid and remember specific locations that can then be revisited automatically, a feature that is particularly useful for tracking a given cell through serial sections.
In most cases, the best strategy is to take plenty of digital images, sort through them after the microscopy session, and perhaps share them with colleagues knowledgeable about EM and about the cellular ultrastructure of interest. Figure 2 shows examples of the complexity of cell structure as viewed in the TEM from samples prepared by conventional chemical fixation, such as the elaborate cytoskeletal arrays in cultured myocytes Figure 2A to the detailed ultrastructure of cellular organelles Figure 2B.
A Actin-myosin cytoskeleton revealed in a cultured cardiomyocyte prepared by conventional chemical fixation. Bar, nm C Golgi membranes in a cultured 3T3 cell prepared by high-pressure freezing and freeze substitution. Bar, nm. D Three-dimensional tomographic model of a forming mitotic spindle from budding yeast. These brief comments concern conventional TEM using thin sections of plastic-embedded material.
There are a number of advanced procedures aimed at better sample preservation or more specialized imaging. Software interfaces capable of automating the tilt series collection and assembling tomograms are available from TEM manufacturers and as freeware Kremer et al.
Tomography is often done with thick sections — nm imaged on an intermediate voltage electron microscope , making it possible to image complex structures in a single section McIntosh et al. Three-dimensional models of cellular structures can then be created from the volume data, such as the forming yeast spindle shown in Figure 2D.
An emerging technology for the preservation of unaltered molecular structure is to prepare samples in vitreous unordered ice without fixation or stain. To image such samples in situ, most cells must be vitrified and then sectioned with cryo-ultramicrotomes to produce frozen sections, a technique referred to as vitreous sectioning Al-Amoudi et al. Frozen sections are imaged using specialized cryo-specimen holders in the TEM.
Electron Microscopy Core Facility - EMBL
The idea is to image the cell in a manner close to its native state. Because the cells have not been fixed or stained, contrast is extremely low, and overall cellular structure can be difficult or impossible to discern. Thus many studies begin with the type of conventional TEM described here and progress to electron tomography, cryo-EM, or cryo—electron tomography to push the resolution and refine the accuracy of a particular structure. In many cases several types of data contribute to the understanding of the structure of interest. TEM is unrivaled in its ability to let one see inside the cell and appreciate the complexity of a chosen structure or organelle.
The techniques are demanding and expensive and may require the expertise of a specialist to accomplish successfully. Experimental design is therefore key; the question to address must be narrowly defined, such that it can be clearly answered with a reasonably sized data set, which in an EM experiment is likely to be small tens of cells. Investigators new to TEM may choose to work with their local EM core facility, or they can establish a collaboration with a group that has expertise in TEM analysis of the cell type, organism, or structure in question.
In any case, there are significant advantages to having the scientist responsible for the experiment be directly involved with the imaging. Familiarity with the system and the experiment will facilitate the search for relevant data, and the process of searching for the best images imparts significantly more information than is captured in images. Barring such an experienced core facility, it is advisable to contact an investigator or core facility that is experienced with one's sample type. Their advice and help with be invaluable and greatly accelerate one's work.
In fact, some core facilities offer sample preparation for outside users and will ship the blocks for sectioning and imaging to the user's local facility. However you get it done, TEM is a powerful and simply fun way to do cell biology. This article is distributed by The American Society for Cell Biology under license from the author s.
Correlative Light and Electron MIcroscopy, Volume 111
Two months after publication it is available to the public under an Attribution—Noncommercial—Share Alike 3. Molecular Biology of the Cell Vol. Janet B. Eileen T. Thomas H. View PDF. Add to favorites Download Citations Track Citations. Abstract Researchers have used transmission electron microscopy TEM to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. Actual user quote in response to particularly beautiful sample. You may embellish with your own words.
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Cryo-electron microscopy of vitreous sections. EMBO J 23 , Cryo-electron tomography on vitrified sections: a critical analysis of benefits and limitations for structural cell biology. Micron 42 , High-pressure freezing for the preservation of biological structure: theory and practice. J Electron Microsc Tech 13 , Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech 3 , Google Scholar Hayat MA Computer visualization of three-dimensional image data using IMOD. J Struct Biol , Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision.
J Cell Biol , Dual-axis tomography: an approach with alignment methods that preserve resolution. High-pressure freezing for preservation of high-resolution fine structure and antigenicity for immunolabeling. Methods Mol Biol , Cryopreparation methods for electron microscopy of selected model systems. Methods Cell Biol 79 , Recent advances in high-pressure freezing: equipment and specimen loading methods. New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol 15 , A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos In: Traffic , 10 The yeast nuclear pore complex: composition, architecture, and transport mechanism.
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