Premium
Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching
Author(s) -
SCHWARTZ CINDI L.,
SARBASH VASILY I.,
ATAULLAKHANOV FAZOIL I.,
MCINTOSH J. RICHARD,
NICASTRO DANIELA
Publication year - 2007
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2007.01794.x
Subject(s) - photobleaching , microscopy , cryo electron microscopy , electron microscope , fluorescence microscope , fluorescence recovery after photobleaching , biophysics , fluorescence , photoactivated localization microscopy , materials science , chemistry , super resolution microscopy , nanotechnology , optics , scanning confocal electron microscopy , physics , biology
Summary Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo‐light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo‐EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo‐LM stage is that photobleaching is slower at cryogenic temperatures (−140°C) than at room temperature.