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.

Figure 1

Advantages of correlative light and electron microscopy (CLEM). (A) Five features of a cell are highlighted (1–5). Blue and green arrows indicate different excitation wavelengths used for light microscopy imaging. Whereas fluorescence imaging allows for the identification only of labeled items (1 and 3), electron microscopy (EM) provides the reference space where all objects are visible (1–5). Due to the limited axial resolution of fluorescence light microscopy (FLM), objects that are closer than 200 nm are blurred into a single spot in the x-y projection (B). CLEM combines two techniques to obtain the highest resolution and information about structures or events occurring within cells.

Figure 2

Example of CLEM using cryosections and the high-data-output CLEM (HDO-CLEM) method. Smooth Russell bodies (SRBs, tubular shape) (A–C) and rough Russell bodies (RRBs, round shape) (D–K)–expressing HeLa cells (Mattioli et al. 2006) have been analyzed by EM and light microscopy. SRBs represent enlarged endoplasmic reticulum–Golgi intermediate compartments, whereas RRBs represent enlarged rough endoplasmic reticulum (RER) compartments. Immunofluorescence staining identifies μ-ΔCH1 (Cy3), a recombinantly expressed mutant immunoglobulin chain accumulated in both SRBs and RRBs. Green labeling refers to calreticulin, a marker for RER. (A) Immunofluorescence labeling for μ-ΔCH1 (Cy3). Confocal laser scanning microscopy (CLSM) optical sectioning poorly resolves the complex tubular architecture of an SRB imaged through the xy middle plane of a three-dimensional (3D) stack; xz axial views along the dotted lines are shown. (B) Transparent surface rendering of the 3D stack shown in A, obtained by MICROSCOBIOJ software. The 3D organization of the SRB tubular structures is not identifiable. The white dot in B corresponds to the cross-point of the dotted lines in A. (C) Immunogold labeling for μ-ΔCH1. Transmission electron microscopy (TEM) image of a 200-nm-thick cryosection of an SRB showing the intricate tubular structure. (D–G) Double immunolabeling on 60-nm or 200-nm (H–K) cryosections of RRBs. (D,H) Immunofluorescence colocalization of calreticulin (Cy3, red) and μ-ΔCH1 (Cy2, green). Nuclei labeled with 4′,6-diamidino-2-phenylindole (blue). Images were collected either by CLSM (D) or by wide-field microscopy (WFM) (H). (G–K) Immunogold labeling colocalization of calreticulin (10 nm) and μ-ΔCH1 (15 nm) of the same sections shown in D and H. (G,K) Higher magnification of the squared areas in E,F and I,J, respectively. Bars: A = 2 μm; C,F,J = 1 μm; D,E,H,I = 5 μm; G,K = 0.5 μm. With permission, Vicidomini et al. Traffic 9:1828–1838, 2008.

Figure 3

HDO-CLEM: Schematic workflow. Pellets from cell cultures are prepared according to the Tokuyasu technique (trimmed block). Ribbons of serial sections are placed on “finder” grids and immunolabeled for light and electron microscopy. A thin layer of methylcellulose is added to the sections to protect them from damage and photobleaching. Grids are next mounted on glass coverslips with glycerol and imaged by WFM and/or CLSM. Once imaging has been completed, grids are extensively washed and stained for EM with uranyl acetate and methylcellulose. Single or serial sections are scanned for areas of interest using grid references and analyzed by standard EM or electron tomography (ET). FLM and TEM-ET images are processed.