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...
|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|