The combination of electron microscopy with transmitted light microscopy (termed correlative light and electron microscopy; CLEM) has been employed for decades to generate molecular identification that can be visualized by a dark, electron-dense precipitate. Merging fluorescence and electron microscopy has proven to be far more difficult, but recent technological advances have enabled the study of biological specimens at high resolution through the introduction of fluorescent probes that are capable of generating contrast for electron microscopy. Among the new reagents available for these studies are genetically-encoded fluorescent proteins and hybrid systems that merge synthetic fluorophores with targeting proteins in living cells. Quantum dots and plant phototropins are also emerging as candidates for CLEM assays.
Bridging fluorescence microscopy and electron microscopy. Histochemistry and Cell Biology 130: 211-217 (2008). An excellent brief overview of correlative light and electron microscopy with a focus on the specific probes used for imaging. Topics include targeting issues, fluorescent proteins, synthetic hybrid systems, quantum dots, and example applications.
Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. Journal of Structural Biology 160: 135-145 (2007). The authors demonstrate the use of cryo-electron tomography of frozen-hydrated biological specimens in correlative microscopy applications. The article discusses instrumentation, software, and sample preparation techniques.
Correlative cryo-light microscopy and cryo-electron tomography: From cellular territories to molecular landscapes. Current Opinion in Biotechnology 20: 83-89 (2009). A discussion of the global nature of multiple imaging modalities spanning a range of spatial scales to examine biological structures at high resolution. The authors discuss cryo-electron tomography, correlative fluorescence microscopy, and time-resolved vitrification.
Markers for correlated light and electron microscopy. Methods in Cell Biology 79: 575-591 (2007). Professor Ellisman and colleagues introduce the complementary nature of light and electron microscopy, and present the principles of using various fluorophores as markers. Also covered are enzymatic-based technology and particle-based methods for protein localization.
Confocal/TEM overlay microscopy: A simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins. Microscopy and Microanalysis 14: 342-348 (2008). Description of a new technique that specifically localizes the position of green and yellow fluorescent proteins within the same cell for imaging in fluorescence and electron microscopy.
Advanced correlative light/electron microscopy: current methods and new developments using Tokuyaso cryosections. Journal of Histochemistry and Cytochemistry 57: 1103-1112 (2009). Alberto Diaspro and co-workers review the major methods currently in use and highlight the latest advances in correlative light and electron microscopy. Among the topics discussed are the application of electron tomography combined with rapid freezing.
Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nature Methods 5: 973-980 (2008). A protocol article that outlines the application of CLEM to examination of living cells using labeling of ultrathin sections with cryo-immunogold techniques. The authors describe the methodology and provide a step-by-step instruction guide.
Photooxidation technology for correlated light and electron microscopy. Journal of Microscopy 235: 322-335 (2009). Photooxidation methods have been established as valuable tools for visualizing cell structures at both the light and electron microscopic level. This article summarizes basic steps of the technology including a discussion on probes and conditions for fine structural localization.
A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. Journal of Microscopy 235: 273-281 (2009). The author reviews high-pressure freezing techniques for CLEM, which is especially important for difficult-to-fix specimens and for the optimal preservation of ultrastructure in samples larger than a few micrometers.
Gaietta, G., Deerinck, T. J., Adams, S. R., Bouwer, J., Tour, O., Laird, D. W., Sosinsky, G. E., Tsien, R. Y. and Ellisman, M. H.
Multicolor and Electron Microscopic Imaging of Connexin Trafficking. Science 296: 503-507 (2002). Roger Tsien teams with Mark Ellisman in an elegant demonstration of CLEM using multicolor fluorescence installed with the FlAsH-ReAsH hybrid system. Gap junction plaque growth was monitored in the optical microscope followed by reduction of ReAsH with diaminobenzidine to generate contrast for electron microscopy.