Researchers used light-sheet microscopy to form 3-D images of the process in primary cortical neurons from embryonic rats.
A new, genetically encoded tag for electron microscopy may revolutionize studies of specific proteins in cells and tissues.
September 1, 2011|
Light microscopy using fluorescent tagging reveals how molecules behave in living organisms, but poor resolution limits how well proteins can be localized. Electron microscopy (EM) provides high resolution, but until now has offered only limited ability to identify specific proteins.
The technique that comes closest to providing high resolution information about protein activity is immunoelectron microscopy, in which gold nanoparticles, readily visible by EM, are bound to protein-specific antibodies. But there’s a trade-off: if antibody labeling is done before fixation, detergents needed to poke holes in the plasma membrane big enough for antibody complexes to enter the cell irreparably damage it. If antibodies are applied after fixation, structures are more intact but the view is superficial as the nanoparticles can’t penetrate very far into the tissue slice.
Roger Tsien, Xiaokun Shu, and colleagues at the University of California, San Diego, engineered a fluorescent Arabidopsis flavoprotein—miniSOG—half the size of green fluorescent protein that can be genetically fused to a wide variety of proteins, transfected into cells, and visualized in the same cells using both light and electron microscopy to more precisely pinpoint proteins. “This method has the potential to revolutionize EM studies throughout biology,” wrote Paul Kaufman from the University of Massachusetts Medical School in his Faculty of 1000 evaluation of the technique (PLoS Biol, 9:e1001041, 2011. Read what researchers are saying).
|METHOD||PREPARATION||WHAT CELL PARTS CAN BE VISUALIZED?|
|MiniSOG||Genetically encode the tag; use regular fixative methods to view under both light microscope and EM||Any part of the cell|
|Immuno-EM||Detergent, which damages the cell structure; fixative limits how far the antibodies penetrate||Top surface of tissue slice; can only visualize proteins for which antibodies exist|
|Metallothionein||Genetically encode metallothionein; soak cellsin solutions of toxic cadmium chloride or gold chloride||Macromolecules or E. coli conditioned to tolerate toxic heavy metals|
|Horseradish peroxidase||Genetically encode HP; use fixative to view. Protein size limits use.||Only proteins that work in the secretory vesicles|