Integrated light and scanning electron microscopy of GFP-expressing cells

Methods Cell Biol. 2014;124:363-89. doi: 10.1016/B978-0-12-801075-4.00017-3.

Abstract

Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes.

Keywords: CLEM; Correlative; Cryo-fluorescence; Green fluorescent protein; ILEM; ILSEM; In-resin fluorescence; Integrated.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism
  • Cell Membrane / ultrastructure
  • Cryopreservation
  • Diglycerides / metabolism
  • Dogs
  • Focal Adhesions / ultrastructure*
  • Green Fluorescent Proteins / biosynthesis
  • HeLa Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Microscopy, Electron, Scanning / methods
  • Microscopy, Electron, Transmission / methods
  • Microscopy, Fluorescence / methods
  • Microtomy
  • Paxillin / biosynthesis
  • Plastic Embedding
  • Pseudopodia / ultrastructure*
  • Recombinant Fusion Proteins / biosynthesis

Substances

  • Diglycerides
  • Paxillin
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins