Single-molecule biophysics research group

University of California, Berkeley



 

PALM (Photo-Activable Light Microscopy)

 

Non-invasive imaging using fluorescent tagging of various cellular organelles in live or fixed cells is a widely used technique in Cell Biology. However, conventional fluorescence microscopy limits us to a spatial resolution of ~200 nm because of diffraction of light. Hence, many sub-diffraction sized organelles and processes are too small to be imaged with high enough resolution. The details obtained by higher resolution imaging can provide important structural details about these organelles in their functioning. We circumvent the limits of diffraction of light by imaging single molecule in 3 dimensions with improved spatial resolution of ~20 nm using Photoactivable Light Microscopy. In this technique, the proteins that are involved formation of cellular organelles are labeled with a photoactivable GFP ( mEos2, Dronpa etc.). Each labeled protein molecule is then stochastically photoswitched or photoactivated and their location in 3D is determined. The final image is reconstructed from all the molecules that localized giving a super-resolved image.

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