Document Detail


Correlative light and electron microscopy (CLEM) as a tool to visualize microinjected molecules and their eukaryotic sub-cellular targets.
MedLine Citation:
PMID:  22588091     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited. On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM), affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy. This powerful technique has been implemented to study many aspects of cell biology. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from ≤5 s up to several hours, depending on the nature of the microinjected sample.
Authors:
L Evan Reddick; Neal M Alto
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Video-Audio Media     Date:  2012-05-04
Journal Detail:
Title:  Journal of visualized experiments : JoVE     Volume:  -     ISSN:  1940-087X     ISO Abbreviation:  J Vis Exp     Publication Date:  2012  
Date Detail:
Created Date:  2012-05-16     Completed Date:  2012-07-20     Revised Date:  2013-08-14    
Medline Journal Info:
Nlm Unique ID:  101313252     Medline TA:  J Vis Exp     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e3650     Citation Subset:  IM    
Affiliation:
Department of Molecular Microbiology, University of Texas Southwestern Medical Center, USA. evan.reddick@utsouthwestern.edu
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
Eukaryotic Cells / chemistry,  cytology*,  ultrasonography
HeLa Cells
Humans
Kidney / chemistry,  cytology,  ultrastructure
Microinjections / methods*
Microscopy, Confocal / methods*
Microscopy, Electron, Transmission / methods*
Microscopy, Fluorescence / methods*
Rats
Subcellular Fractions / chemistry,  ultrastructure
Grant Support
ID/Acronym/Agency:
AI083359/AI/NIAID NIH HHS; R01 AI083359/AI/NIAID NIH HHS
Comments/Corrections

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