CLEM in the Classroom
Protocols for Undergraduate Courses
- CLEM in the Classroom - Protocols for Undergraduate Courses
- Fig. 1: CLEM of immunolabeled sections. (a) Sample preparation and labeling strategy (using e.g. rabbit primary antibodies). (b) Workflow, labeling of resin sections. (c) Workflow, labeling of Tokuyasu-cryosections. (d) Workflow, labeling of resin section for use as dried demo samples.
- Fig. 2: Rhodopsin in mouse retina sections. (a,b) Overview FLM (a) and TEM (b), the square indicates the region displayed in (c). (c) Overlay fluorescence and TEM, outer segments (os) of rod photoreceptor cells are labeled; cc, connecting cilium; is, inner segment. Two regions of interest (ROI 1,2) are selected for analysis at higher magnification. (d,e) ROI 1, (f,g) ROI 2, immunogold labeling of rhodopsin in the membrane stacks of outer segments. Labeling of Lowicryl K4M sections through mouse retina (RPE removed) using mouse anti-rhodopsin, rabbit-anti-mouse, goat-anti-mouse Alexa488, and protein A 10 nm gold.
- Fig. 3: Actin in mouse intestine sections. (a,b) Overview FLM (a) and TEM (b). One crypt (cry, dashed line) is selected for further analysis; mu, smooth muscle; sub, submucosa. (c) The crypt indicated as ROI (dashed lines) in a,b, overlay fluorescence/TEM. Intestinal cells including enterocytes (ent), Paneth cells (pac) and goblet cells (goc) are visible. The square indicates the region displayed in d. (d) Lumen (lu) close to the base of the crypt. Apical domains of intestinal cells with terminal web (tw) and microvilli (mv) are visible. The square indicates the region displayed in e. (e) Apical surfaces of intestinal cells with junctional complexes (tj, tight junction; aj, adherens junction; d, desmosome). Labeling of Lowicryl K4M sections with mouse anti-actin, rabbit-anti-mouse (bridge), goat-anti-mouse Alexa488, and protein A 10 nm gold.
- Fig. 4: Cryosections through mouse retina after transplantation of GFP-labeled cone-like cells. Labeling with rabbit anti-GFP, goat-anti-rabbit Alexa488 and protein A 10 nm gold, and DAPI. (a) FLM, some GFP-positive cells are integrated. (b) TEM-micrograph of the same area, onl, outer nuclear layer; is, inner segments; os, outer segments. (c) Overlay FLM/TEM, gold labeling (not visible at this magnification) is indicated by black dots.
- Fig. 5: K4M-section through mouse retina, Rhodopsin-GFP transgenic. Anti-GFP staining with fluorescent markers (a) and Nanogold + silver enhancement (b,c). Signal can be seen in the outer segments of rod photoreceptors. FLM- and TEM imaging was done with contrasted (10 min 2% uranyl actetate) and dried samples. Silver enhancement helps to achieve a good balance between fluoescence and EM-contrast, but the staining works also with protein A gold.
Correlative light and electron microscopy (CLEM) combines the versatility of fluorescence microscopy (FLM) with the high spatial resolution of electron microscopy (EM). Whereas many CLEM-protocols are complex and/or need sophisticated instrumentation, CLEM of immunolabeled ultrathin sections is an established, versatile and fast method for cell and tissue analysis. It is suitable for teaching at various levels in undergraduate courses.
Correlative Light and Electron Microscopy (CLEM)
In the life sciences FLM is an indispensable tool to visualize cell types, cellular organelles, protein distribution, or signaling events in cells and tissues. However, the resolution of FLM is limited by diffraction (20-200 nm) , and signal detection is limited to fluorescently labeled structures. EM, on the other hand, reveals subcellular details of both labeled and unlabeled structures, but can be applied only to fixed samples, and labeling is performed using only a handful of different electron-dense markers. Correlative light electron microscopy (CLEM) combines the versatility of FLM with the high spatial resolution of the EM to bridge the resolution gap between the two imaging modalities and combine the best of both worlds.
In vivo FLM can be performed in cell culture systems, small organisms, or some embryos [2-5]. After imaging these samples are fixed, embedded and serially sectioned to retrace the region of interest (ROI) in the transmission electron microscope (TEM) [2-4]. Although this approach allows the ultrastructural analysis of ROIs with a known history (through life-cell imaging), it is time consuming and limited to only one ROI per sample.
For the correlative analysis of larger samples such as tissues or larger embryos the specimens have to be fixed, embedded, and sectioned before CLEM. Tissue analysis occurs on resin- or Tokuyasu-cryosections [6-13]. To facilitate the correlation of FLM and EM data, the sections can be immunolabeled using fluorescence and gold-labeled secondary markers [6,11]. This approach is versatile, offers several ROIs per section, thin high-resolution FLM-samples (50-100 nm), and the opportunity to label many different antigens on sections from a single sample.
CLEM of Immunolabelled Ultrathin Sections in Undergraduate Courses
Since many CLEM-protocols are time-consuming and complex or depend on elaborate technical skills or sophisticated instrumentation [2-4; 14-18], they are usually not the subject of undergraduate courses.
CLEM of immunolabeled sections, however, is relatively easy to perform using ultrathin sections for on-section labeling and standard fluorescence and electron microscopes for imaging [6,11]. The labeling procedure can be performed in half a day and in case of time limitations dry demo samples can be prepared in advance.
In our courses, FLM and EM are performed on the very same ultrathin sections mounted to meshed EM-grids. The general labeling protocol and the workflows for resin sections, cryosections or demo samples is shown in figure 1. In brief, tissue samples were fixed and processed for immunogold labeling. Ultrathin sections, mounted on grids, were stained with primary antibodies and protein A gold followed by Alexa488-labeled secondary antibodies which attach to binding sites that are not occupied with protein A gold. This way, the sections are simultaneously labeled with fluorescent and gold markers . Finally, sections are counterstained with DAPI, mounted on glass and analyzed at the FLM. Areas of interest are selected and imaged, before the sections are stained with uranyl acetate for EM-inspection.
Two student course samples are shown in figures 2 (mouse retina with stained rod photoreceptors) and 3 (mouse intestine with stained actin-cytoskeleton). Sections are cut from 6 year old K4M-blocks without significant loss of signal intensity over the years. The samples demonstrate typical applications for CLEM, such as the illustration of cell types with specific markers (fig. 2) or of subcellular structures such as the cytoskeleton (fig. 3). Both experiments were performed during a 1-week basic EM-course in our master program.
Another typical application of CLEM is the identification and analysis of rare cell populations in complex tissues, e.g. via GFP expression [e.g. 10-12] (fig. 4). In this case the CLEM was performed on immunolabelled Tokuyasu-cryosections. Here, the staining procedure is more complex (fig. 2c)  but this example nicely illustrates the strength of section-CLEM for tissue analysis.
Finally, for demonstrations of the basic principle of CLEM ready-to-go samples can be prepared in advance using protocols designed for section-CLEM with integrated microscope systems, where both FLM and EM are performed in the vacuum of the electron microscope [15, 17]. These demo samples were immunolabeled, contrasted and dried before imaging (fig. 1d, fig. 5). If kept in the dark, the fluorescence is stable for more than 6 months and can be used repeatedly for demonstrations.
Taken together, CLEM of immunolabeld tissue sections is an effective method to teach the principles and benefits of correlative analysis in the life sciences.
We would like to thank the Ader-group at the CRTD for the retina samples which are used in our EM-courses, the European Fund for Regional Develoment (EFRE), and the Deutsche Forschungsgemeinschaft (DFG, FZT 111 CRTD, Cluster of Excellence) for funding, and the students of the master courses „Regenerative Biology and Medicine“ and „Biology“ at the TUD for providing nice CLEM-images every year.
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Dr. Thomas Kurth
DFG-Center for Regenerative Therapies Dresden (CRTD) and BIOTEC Center,
Electron Microscopy and Histology Facility