Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching

Cindi L. Schwartz, Vasily I. Sarbash, Fazoil I. Ataullakhanov, J. Richard Mcintosh, Daniela Nicastro

Research output: Contribution to journalArticle

120 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)98-109
Number of pages12
JournalJournal of Microscopy
Volume227
Issue number2
DOIs
StatePublished - Aug 1 2007

Keywords

  • Correlative light and electron microscopy
  • Cryo-electron tomography, cryo-light microscopy
  • Green fluorescent protein
  • Microtubules

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology

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