Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Abstract

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

“…In the last 10 years there has been significant interest in imaging fluorescent proteins in semithin sections (for review, see Cortese et al, 2009). Luby-Phelps and colleagues (2003) first described a method for retaining GFP fluorescence after fixation and resin embedding, but their method has not seen widespread application.…”

The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms

“…A second alternative approach by which the subcellular location of fluorophores can be made EM-visible is by photoconversion of diaminobenzidine (DAB) [40]. This approach uses fluorophores to photooxidize DAB, which results in precipitates after reaction with osmium.…”

Advances in high-resolution imaging – techniques for three-dimensional imaging of cellular structures

“…CLEM combines the advantages of these two techniques to allow direct examination of fluorescence localization within the high-resolution landscape of EM images. Recent advances in probe development, sample preparation and instrumentation are making CLEM more widely accessible (Darcy et al, 2006;Agronskaia et al, 2008;Cortese et al, 2009;Plitzko et al, 2009;Briegel et al, 2010;Bos et al, 2011;Bushby et al, 2011).…”