Correlative Light and Electron Microscopy
Workflow in an Electron Microscopy Facility
- Correlative Light and Electron Microscopy - Workflow in an Electron Microscopy Facility
- Fig. 1: Correlative light-electron microscopy of cells containing a GFP-tagged subgenomic replicon. (A) Epi-fluorescence microscope image of live cells containing a subgenomic replicon with a GFP-tagged NS5A. Huh7-Lunet cells were transfected with replicon RNA and seeded onto patterned sapphire discs. Twenty-four hours later cells were analyzed by fluorescence microscopy and immediately processed for EM. (a) Fluorescence image; (b) and fluorescence images of the cell of interest: (c) merge of bright field and fluorescence images of the same area. Coordinates etched onto the surface of the sapphire disc were used to record the position of the selected cells. (B) Overlap of the fluorescence image and a low magnification EM micrograph of the cell highlighted and enlarged in panel A. (C) Higher magnification images of the region marked with the white box in panel B. The NS5A-GFP signal corresponds primarily to LDs and accumulations of DMVs residing in close proximity of them. Image courtesy of Ines Romero-Brey and Ralf Bartenschlager.
- Fig. 2: Correlative light-electron microscopy work flow using live cell imaging. (A) Image of a sapphire disc used to high-pressure freeze cells, with a finder grid pattern carbon evaporated onto the surface. Scale bar represents 300 µm. (B) Bright-field image of two cells growing on a patterned sapphire disc expressing a cell line with lamin-gfp and H2B m-Cherry. In the background is the pattern of the letter R on the sapphire disc that will be used later to find back the cells of interest. (C) Epi-fluorescence image of the two cells of interest shown in the GFP channel (470 nm). (D) Epi-fluorescence image of the two cells of interest with the GFP and m-Cherry channels overlaid (470 and 590 nm). (B)-(D) Scale bar represents 20 µm. (E) Transmission electron micrograph of a 60 nm section through one of the cells of interest. White arrows are pointing to nuclear pore complexes labeled with an antibody against nucleoporins attached to a 10 nm protein-A gold particle. The scale bar represents 500 nm.
Robust sample preparation is key to any multiuser, high-end electron microscopy facility. At the EMBL our facility is permanently hosting 10 to 15 projects in parallel, and provides service to approximately 50 users per year. Each of these users face a number of different challenges when it comes to sample preparation and have different levels of experience. Our goal is to assist by standardizing methods and developing a toolbox to help projects become feasible and more efficient.
Strategies to Capture and Study Dynamic Events
In recent years correlative light and electron microscopy (CLEM) has become a highly fashionable method using fluorescent markers to locate the region of interest (ROI) by light microscopy (LM) and combining it with the high-resolution data achieved from the electron microscope. Our facility is using a number of different CLEM methods and has played an active role in developing strategies to capture and study dynamic events at high-resolution.
Finding a rare object or event in a cell culture or in an organism to later perform ultrastructural analysis at the exact same position is not trivial. One method we are using is fluorescent imaging after mild fixation. Cells expressing fluorescent-tagged proteins are seeded onto dishes having a glass coverslip with a gridded pattern on them (MatTek, Ashland, MA, USA). Once at the right confluency, the cells are fixed with a mild fixative and then imaged without haste on a fluorescent microscope. The correlation is performed by recording the position of the cell of interest relative to a system of coordinates pre-existent onto the cultured surface or by adding landmarks while performing the LM imaging . Following imaging, the cells are fixed again with glutaraldehyde and processed further until they are embedded into an epoxy resin. The advantage is that the landmarks are visible on the resin block and finding back the cell of interest becomes an easier task. A second method used to perform ultrastructural analysis is live-cell imaging followed by fast cryo-fixation  (fig.
1, 2). Cells are seeded onto sapphire disks having a pattern either etched on or carbon evaporated onto the surface. Once frozen, the cells are freeze-substituted and embedded into a resin block where the pattern is again visible on the surface.
Studies on Larger Samples
For correlative studies on larger specimens such as starfish oocytes or nematodes, we have implemented specific techniques where the sample is processed inside micro-capillary tubes  or embedded into agarose . Live-cell imaging becomes easier as the sample is completely immobilized. One can perform time-lapse microscopy and cryo-fix the sample at the time point of interest. Samples are then freeze-substituted and embedded into a resin of choice. Using targeted ultramicrotomy, the LM map of the sample is then used to selectively section through the region of interest.
Not only can the fluorescence be imaged on living specimens but also after fixation and sample preparation for EM, as demonstrated in 2011 by Kukulski et al. . This method allows one to directly visualize fluorescent proteins on a thick section. The specimen is high pressure frozen, freeze substituted and embedded into a low-temperature embedding resin. Once sections are made they are imaged on the LM, which allows for a more rapid screening of the ROI in several cells. By using fluorospheres as bi-functional fiducials (fluorescent and electron-dense), one can relocate the points of interest in the LM and then in the EM with high precision. Furthermore, this in combination with electron tomography provides precise correlation together with information from the complete 3D volume. As with any method, there are a few things one should consider beforehand. The fluorescent signal must be bright to start with and once the sections are cut, imaging should be done within a 24-hour period. The blocks must be stored in the dark and can be used for several weeks depending on how they are stored.
Electron Microscopy Core Facility
In an environment such as EMBL, our facility is at the interface between methodological development and providing service to our community of users. It is therefore playing a key role in making these methods accessible to a large number of researchers. The Electron Microscopy Core Facility has close collaborations with research groups from diverse research areas using correlative methods such as Cell Biology, Developmental Biology and Structural Biology. The groups of John Briggs, Péter Lénárt, Rainer Pepperkok and Yannick Schwab, amongst several others within EMBL, play a key role in this transfer. Similar benefits come from interactions outside of the EMBL such as with the groups of Thomas Mueller-Reichert at the Technical University in Dresden, Germany and of Ralf Bartenschlager in the Molecular Virology Department at the University of Heidelberg, Germany.
Over the past decade, many laboratories have been developing original approaches in CLEM. Often tedious, and always relying on the skills of trained experts, these powerful techniques now have the potential to be widely spread. As they often represent the only solution to address specific questions on biological systems they are expected to play a key role in many research areas. As a consequence, any group that has invented such new approaches is facing an increasing demand for collaborations that are hard to satisfy. It is therefore logical to ask core facilities to assimilate the methods, adapting them to a large diversity of applications. The Electron Microscopy Core Facility at EMBL has achieved this step and is now able to offer to its users CLEM methods spanning a wide spectrum of sample types and biological questions.
RSM would like to thank the Electron Microscopy Core Facility at the EMBL, Claude Antony, Uta Haselmann-Weiss and Inés Romero-Brey for their support and help with method development and Yannick Schwab for critical reading of the manuscript.
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 Romero-Brey I. et al.: PLoS Pathog. 8 (12), e1003056 (2012)
 Joseph-Strauss D. et al.: Dev Biol. 365 (2), 445-57 (2012)
 Kolotuev I. et al.: Biol Cell. 102 (2), 121-32 (2009)
 Kukulski W. et al.: Methods Cell Biol. 111, 235-57 (2012)
Rachel Santarella-Mellwig (corresponding author via e-mail request)
European Molecular Biology Laboratory
Electron Microscopy Core Facility