Advertisement
RayBiotech
RayBiotech

Deep Tissue Treatment

A new, genetically encoded tag for electron microscopy may revolutionize studies of specific proteins in cells and tissues.

By | September 1, 2011

image: Deep Tissue Treatment

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).

 

STATS TALK
METHODPREPARATIONWHAT CELL PARTS CAN BE VISUALIZED?
MiniSOGGenetically encode the tag; use regular fixative methods to view under both light microscope and EMAny part of the cell
Immuno-EMDetergent, which damages the cell structure; fixative limits how far the antibodies penetrateTop surface of tissue slice; can only visualize proteins for which antibodies exist
MetallothioneinGenetically encode metallothionein; soak cellsin solutions of toxic cadmium chloride or gold chlorideMacromolecules or E. coli conditioned to tolerate toxic heavy metals
Horseradish peroxidaseGenetically encode HP; use fixative to view. Protein size limits use.Only proteins that work in the secretory vesicles
 

Advertisement

Add a Comment

Avatar of: You

You

Processing...
Processing...

Sign In with your LabX Media Group Passport to leave a comment

Not a member? Register Now!

LabX Media Group Passport Logo

Follow The Scientist

icon-facebook icon-linkedin icon-twitter icon-vimeo icon-youtube
Advertisement
RayBiotech
RayBiotech

Stay Connected with The Scientist

  • icon-facebook The Scientist Magazine
  • icon-facebook The Scientist Careers
  • icon-facebook Neuroscience Research Techniques
  • icon-facebook Genetic Research Techniques
  • icon-facebook Cell Culture Techniques
  • icon-facebook Microbiology and Immunology
  • icon-facebook Cancer Research and Technology
  • icon-facebook Stem Cell and Regenerative Science
Advertisement
Hamamatsu
Hamamatsu
Advertisement
NeuroScientistNews
NeuroScientistNews
Life Technologies