Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers

Methods Cell Biol. 2012;111:235-57. doi: 10.1016/B978-0-12-416026-2.00013-3.

Abstract

The application of fluorescence and electron microscopy to the same specimen allows the study of dynamic and rare cellular events at ultrastructural detail. Here, we present a correlative microscopy approach, which combines high accuracy of correlation, high sensitivity for detecting faint fluorescent signals, as well as robustness and reproducibility to permit large dataset collections. We provide a step-by-step protocol that allows direct mapping of fluorescent protein signals into electron tomograms. A localization precision of <100 nm is achieved by using fluorescent fiducial markers which are visible both in fluorescence images and in electron tomograms. We explain the critical details of the procedure, give background information on the individual steps, present results from test experiments carried out during establishment of the method, as well as information about possible modifications to the protocol, such as its application to 2D electron micrographs. This simple, robust, and flexible method can be applied to a large variety of cellular systems, such as yeast cell pellets and mammalian cell monolayers, to answer a broad spectrum of structure-function related questions.

MeSH terms

  • Acrylic Resins / chemistry
  • Animals
  • Cells, Cultured
  • Cryopreservation
  • Electron Microscope Tomography*
  • Fiducial Markers*
  • Fluorescent Dyes / chemistry
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / chemistry
  • Image Processing, Computer-Assisted
  • Microscopy, Fluorescence
  • Microtomy
  • Plastic Embedding
  • Yeasts / ultrastructure

Substances

  • Acrylic Resins
  • Fluorescent Dyes
  • Green Fluorescent Proteins
  • Lowicryl HM-20