Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching.
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 degrees C) than at room temperature.
Schwartz
Cindi L
CL
Boulder Laboratory for 3D Electron Microscopy of Cells, University of Colorado, Department of Molecular, Cellular, and Developmental Biology, Boulder, CO, USA.
Sarbash
Vasily I
VI
Ataullakhanov
Fazoil I
FI
McIntosh
J Richard
JR
Nicastro
Daniela
D
D018929:Cell Culture Techniques; D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D017110:Freeze Substitution; D006652:Histological Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D038761:Photobleaching; D016610:Tissue Embedding