CLEM on Ultrathin Sections

From Micro- to Nanoscale

  • CLEM on Ultrathin Sections - From Micro- to NanoscaleCLEM on Ultrathin Sections - From Micro- to Nanoscale
  • CLEM on Ultrathin Sections - From Micro- to Nanoscale
  • Fig. 1: CLEM of cell-cell junctions in Tokuyasu cryo-sections through intestinal epithelial cells. Labeling of consecutive but separate sections. Left: Double-immuno-FLM of tight and adherens junction markers (ZO-1, red; β-catenin, green). Right: TEM-micrograph of a cryo-section labeled with anti- β-catenin/ protein A 10 nm gold, fluorescence overlay: tight junction (red), adherens junction (green).
  • Fig. 2: CLEM of Tokuyasu cryo-sections through the retina of a transgenic mouse expressing rhodopsin-GFP. (a) GFP-fluorescence in the outer segments (os); gcl, ganglion cell layer; inl, inner nuclear layer; is, inner segments, onl, outer nuclear layer. (b) Immunofluorescence after labeling with anti-GFP and Alexa555-coupled secondary antibodies, nuclei counterstained with DAPI. (c) Immunogold labeling of rhodopsin-GFP in the membrane discs of an outer segment.
  • Fig. 3: CLEM of a single resin section through wild-type mouse retina after transplantation of photoreceptor precursor cells (PPCs) into the subretinal space. (a) Sample preparation and on-section labeling strategy, modified from [10]. (b-d) Identification of a GFP-labeled PPC (arrowheads) at the FLM (b,c) and the TEM (d), the asterisks indicate a dirt particle, modified from [9].
  • Fig. 4: CLEM of the GFP-labeled PPC that was selected in figure 3. (a,b) The PPC (arrowheads) integrated into the photoreceptor layer of the host animal, from [9]. (c) The area indicated in (a) at higher magification, the integrated PPC formed a proper outer segment with parallel membrane stacks full of Rhodopsin-GFP, immunogold labeling highlighted in red. The TEM image provides the reference space with additional information such as the connecting cilium of the integrated PPC (cc), inner segments and non-labeled wild-type outer segments of the host (asterisks).

Correlative light and electron microscopy (CLEM) combines the versatility of fluorescence microscopy (FLM) with the spatial resolution of transmission electron microscopy (TEM). For the analysis of tissues single resin or cryo-sections are incubated with fluorochrome- and gold-labeled probes. Areas of interest are selected at the FLM, and analyzed in the TEM at high resolution. This way, fluorescence is directly correlated to subcellular structures and/or corresponding immunogold signals.

Why Correlative Light and Electron Microscopy (CLEM)?

Fluorescence Light Microscopy (FLM) is one of the most powerful tools in cell biology and many different fluorescent markers are available to visualize cellular components, protein distribution, signaling events, or biochemical reactions in living cells. However, the resolution of FLM is limited by diffraction. Moreover, only labeled structures can be imaged, whereas all unlabeled structures in the vicinity, which together form the so-called reference space, remain undetected. Very often, even the details of labeled structures remain obscure. Although super-resolution microscopes have shifted the diffraction barrier to 20-60 nm [1], they did not solve the issue of the invisible reference space. TEM, on the other hand, reveals subcellular details of both labeled and unlabeled structures, but it is limited to fixed and sectioned samples, and only a few electron dense markers are available. Correlative light electron microscopy (CLEM) combines the versatility of FLM with the high spatial resolution of TEM to visualize the totality of subcellular structures.

CLEM on Ultrathin Sections

Correlation of in vivo imaging and EM has been performed in many cell culture systems [2, 3]. Beyond cell culture, however, this approach is limited to very small specimens and embryos [4]. For the correlative analysis of larger samples such as tissues, the specimens have to be fixed, embedded, and sectioned before the correlation. The CLEM analysis of tissues is performed on resin- or on Tokuyasu cryo-sections. The advantages of this approach are: (1) Many areas of interest can be analyzed and statistically evaluated; (2) thin sections are excellent FLM-samples, because the fluorescence is emitted from a very thin optical plane (50-200 nm); (3) the method is easy and widely applicable; (4) many different antigens can be analyzed on sections from a single sample.

One option to perform CLEM is to lable separate sections which were cut from the same tissue block (figs.

1 and 2). These sections are mounted and processed independently for FLM and EM [5, 6]. This approach is useful for target structures that are abundant and of regular shape, so that the correlation of FLM and TEM-data of individual structures is not necessary (e.g. many cell organelles, junctions between epithelial cells, fig. 1, GFP-labeled cell types in transgenic animals, fig. 2). In fact, any immuno-EM experiment benefits from CLEM, because the fluorescence helps to identify target areas and to judge the quality of labeling before TEM-analysis.

In principle, fluorescent signals can be correlated to labeled or unlabeled structures in the TEM. In figure 1, for example, the adherens junctions were labeled with fluorescent (red) and with gold markers (10 nm particles), whereas the tight junction is labeled only with a fluorophore-coupled antibody (green).

Alternatively, CLEM is performed on the very same ultrathin section [7-10]. This approach is necessary when the structures of interest are either very small (vesicles, granules, small organelles), and may not be imaged on two different sections, even if consecutive sections are analyzed. It is also helpful when rare cell populations are the subject of interest (stem cells, transplanted cells, etc.). Figures 3 and 4 illustrate this on resin sections through wild-type mouse retina after transplantation of photoreceptor precursor cells (PPCs) from transgenic mice expressing GFP-labeled rhodopsin (Rho-GFP) [9]. The labeling protocol is depicted in figure 3a [10]. In brief, retina samples were fixed and processed for immunogold labeling. Ultrathin sections were mounted on finder grids, and stained with anti-GFP and protein A gold. After that, the samples were incubated with 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. Several alternative approaches using different combinations of markers are available [10]. Finally, sections are counterstained with DAPI, mounted on glass and analyzed at the FLM. Areas of interest are selected and imaged. In our example most of the transplanted PPCs are found in a coherent group of cells in the subretinal space (fig. 3b) and some PPCs integrate into the host photoreceptor layer (fig. 3c). After FLM, the grid was stained with uranyl acetate and analyzed at the TEM. One of the integrated PPCs (arrowhead in figs. 3c,d and 4a,b) was selected and imaged at high resolution (fig. 4c), providing additional structural information of the PPC that developed a decent outer segment with Rho-GFP filled membrane discs. In the reference space (unlabeled black surroundings in fig. 4b) inner segments and unlabeled outer segments of the host are visible. Therefore, CLEM on tissue sections reveals protein expression data and fine structural information from the micro- to the nanoscale.

Outlook and Further ­Challenges

Among the recent developments are 3D-CLEM [7], CLEM using super-resolution microscopes and TEM [11,12], and the advent of integrated systems for CLEM, such as the Tecnai with iCorr from FEI. Such a combined FLM/TEM system poses new challenges to sample preparation and on-section labeling. In the protocol described above, FLM and contrast generation for EM are separated processes. In an integrated system fluorescence and electron microscopy are both performed on dry samples in the vacuum of the EM column. To achieve a decent compromise between fluorescence intensity and sample contrast under these conditions is a challenging task [13, 14].

Acknowledgements
We would like to thank John Wilson for Rho-GFP mice and the European Fund for Regional Develoment (EFRE), the Deutsche Forschungsgemeinschaft (DFG, FZT 111 CRTD, Cluster of Excellence), and ProRetina e.V. for funding.

References
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[8] Kukulski W. et al.: J. Cell Biol. 192, 111-119 (2011)
[9] Eberle D. et al.: PLOS One 7, e46305 (2012)
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[14] Karreman M.A. et al.: J. Histochem Cytochem 61, 236-247 (2013)

Authors
Dr. Thomas Kurth
(corresponding author via e-mail request)
Susanne Kretschmar
Dr. Dominic Eberle
Dr. Marius Ader
TU Dresden,
DFG-Center for Regenerative Therapies Dresden (CRTD),
Dresden, Germany 

Contact

University of Technology Dresden
Fetscherstraße 105
01307 Dresden
Germany

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