KENBIKYO Vol.49▶No.3 2014
â– Lectures

PALM, Superresolution Fluorescence Microscopy Circumventing the Diffraction Limit of Light, to Explore Minute Structures of Cells

Susana Rocha, Herlinde De Keersmaecker, Mitsuhiro Abe, Hiroshi Uji-i, Johan Hofkens, Toshihide Kobayashi, Atsushi Miyawaki and Hideaki Mizuno

Abstract: Fluorescence microscopy is a common tool in the field of the life science for specific labeling and observation of target molecules. Methods of non-invasive staining have been established by ectopic expression of fluorescence proteins, with which the fluorescence microscopy has been applied to live-cell imaging. Superresolution microscopy circumventing the diffraction limit of light has also been developed recently. Here we focus on one of the superresolution microscopic modalities, photoactivated localization microscopy (PALM). For PALM, a target molecule is labeled with a photoswitchable fluorescent protein. Using a stochastic switching of the fluorescent protein, spatially overlapping molecules can be temporally isolated, and the coordinates of respective molecules are calculated from fitting of the signals to the two-dimensional Gaussian function. With PALM, the target molecules can be observed with the resolution of tens of nm. By staining cells with recently developed probes, PALM is also applicable for the imaging of membrane rafts. It can also be used for the single particle tracking in living cells to analyze molecular dynamics.

Key words: superresolution microscopy, PALM, photoswitchable fluorescent protein, membrane raft, single molecule tracking