Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Abstract

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.

Introduction

Correlative light and electron microscopy (CLEM) encompasses all microscopic techniques that aim at imaging the same area in a specimen by using photons and electrons sequentially as the image-forming sources. The combination of these two imaging modalities offers specific advantages. Thus the relatively low-magnification range of light microscopy (LM) is ideal for locating areas of interest for subsequent high-resolution imaging by transmission electron microscopy (TEM). For comparison, typical fields of view are 50 by 50 μm with a resolution of 200 nm for LM, and 4 by 4 μm with a resolution of 1 nm for TEM. Performing the search for areas of interest directly by TEM can be as laborious as finding the proverbial needle in a haystack due to the higher magnification range at which TEM operates (Koster and Klumperman, 2003). This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in 2D search mode due to the superimposing of 3D structural information in the 2D projection image. For cryoelectron microscopy the challenge to locate an area of interest is even greater, since the low-dose requirements prohibit extensive searching, and the low contrast provides little visible morphological information in the 2D image (Braet et al., 2007, McIntosh et al., 2005, Steven and Aebi, 2003). An additional important advantage of LM is that it allows for the imaging of dynamic processes in live cells, thereby establishing a history of the cells and, as a consequence, adding considerably to the interpretational gain of subsequent ultrastructural analyses by TEM or electron tomography (Svitkina and Borisy, 1998).

Although CLEM techniques have been available and applied in many research projects within the life sciences for several decades, they have not yet become mainstream tools despite the obvious benefits outlined above. One reason for CLEM remaining on the fringe of TEM and electron tomography applications is that the currently available protocols often appear highly specialized and therefore suitable only for addressing a limited number of questions. Another reason is that many of the existing procedures seem arduous, cumbersome, and requiring special skills. Several factors contribute to these current drawbacks. First of all, there is no golden standard yet for an intermodal coordinate system that permits easy retrieval at the TEM level of an area of interest preselected by LM. Moreover, the transition from LM to TEM often involves additional processing such as embedding and ultra-thin sectioning, which can lead to changes in orientation of the sample (Biel et al., 2003, Giepmans et al., 2005, Mironov et al., 2000). Secondly, there are considerable differences between LM and TEM with respect to the preparation of biological samples, and their unification often implies a compromise between stainability and ultrastructural preservation (Giepmans et al., 2006). For instance, osmium tetroxide, a commonly used postfixative that generates contrast for TEM on plastic-embedded material, is incompatible with most LM staining or labeling procedures. Thirdly, different detection systems are employed to localize specific molecules by LM or by TEM (Giepmans et al., 2006, Grabenbauer et al., 2005). Although progress is being made in the development of multimodal probes (Corstjens et al., 2005, Deerinck et al., 2007, Giepmans et al., 2005, Najlah and D’Emanuele, 2006, Powell et al., 1998, Takizawa and Robinson, 2000), there are no routine methods yet for the correlated localization of biomolecules.

It is, however, common practice in most electron microscopy laboratories to cut semi-thin (0.3–0.5 μm) plastic sections adjacent to ultra-thin (50–70 nm) sections; the semi-thin sections are stained with toluidine blue to provide contrast for light microscopic examination (McNary et al., 1964). This procedure is very useful to provide a general overview of the sample area as a prelude to the electron microscopic investigation of the ultra-thin sections. However, due to the considerable thickness of the semi-thin sections, it is not possible to browse for fine details such as subcellular structures, or even whole cells, as they may not be present anymore in the adjacent ultra-thin sections. The ultra-thin sections cannot be stained directly since they absorb too little toluidine blue (or similar chromophores) to generate sufficient contrast (Jones et al., 1982). Much weaker signals can be detected when fluorescent stains are used instead of chromophores (McNary et al., 1964). Biel et al., developed a method for the fluorescent staining of resin-embedded skin tissue by adding fluorescent dyes, such as acridine orange and safranin O, to the substitution medium during freeze-substitution (Biel et al., 2003, Pfeiffer et al., 2003). The fluorescent signals are then recorded by imaging in the specimen block using 3D confocal laser scanning microscopy, and subsequent electron micrographs acquired from ultra-thin sections are matched with an optical slice from the 3D volume (Biel et al., 2003).

The present paper reports our efforts to develop more universally applicable protocols for CLEM. We focused on feasible procedures that allow for the fluorescent staining of thin sections of resin-embedded cells and tissues. These stains provide landmarks to facilitate the browsing of complex tissues, and also enable the detection of rarely occurring features that would otherwise be extremely difficult to find. Finally, we propose the use of electron tomography with a minimum number of tilt angles to generate 2D images with improved contrast and satisfactory ultrastructural detail despite the lack of osmification.