In correlative light and electron microscopy, the discerning power of fluorescence microscopy (FM) is combined with the ultra-high resolution of electron microscopy (EM). We fluorescently label protein complexes in cells, with the purpose of facilitating their identification in transmission electron tomograms. Our imaging strategy is to perform cryogenic selected volume tomography, continuously keeping the specimen in a vitreous state. To this end, we are developing a cryogenic workflow in collaboration with FEI, consisting of three microscopes: CorrSight light microscope (LM), Scios Dualbeam scanning electron microscope (SEM) with a focused ion beam (FIB) and Tecnai Arctica cryo–transmission electron microscope (TEM). The focus of my presentation is on the integration of the CorrSight light microscope into the workflow (1). This translates to performing cryogenic light microscopy and correlating the images with EM for guided ion beam milling and tomography (2).

The data we collected from cryogenic fluorescence microscopy of mammalian and bacterial cells on TEM grids shows great promise for complementing EM (Fig. 1). This is substantiated by alignment of LM and SEM images utilizing FEI MAPS software, allowing for fluorescence based navigation during SEM imaging (Fig. 2). My future efforts will focus on improving localization precision and correlation accuracy, to guide FIB-milling of sub-cellular structures in the SEM. FIB-milling is used to fabricate thin lamellae in cells, making them suitable for cryo-TEM. Our results indicate that tomograms can be recorded from lamellae using a 200kV cryo-TEM, but vitrification of the specimen is essential. The ultimate goal of our workflow is to obtain structural insight of the Type 7 Secretion System (T7SS), formed by the ESX-1 machinery. To this end, we will perform tomography of mycobacterial membranes inside professional antigen-presenting cells. The T7SS is an important part of the mycobacterial protein weaponry, as it mediates virulence and phagosomal escape (4, 5).

References

1. Arnold, J., J. Mahamid, A. de Marco, J.-J. Fernandez, T. Laugks, et al. 2016. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. Biophysical Journal. 110: 860.

2. Mahamid, J., S. Pfeffer, M. Schaffer, E. Villa, R. Danev, et al. 2016. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science. 351: 969.

3. K.V. Korotkov, and W. Bitter. 2014. Take five — Type VII secretion systems of Mycobacteria. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 1843: 1707.

4. Smith, S. 2004. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Molecular Microbiology. 51: 359.

5. van der Wel, N., D. Hava, D. Houben, D. Fluitsma, M. van Zon, et al. 2007. M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells. Cell. 129: 1287.

Figures:

Figure 1: Cryogenic light microscopy of mammalian cells on EM grids. The specimen contains J774 macrophages labeled with LysoTracker Red, cultured on golden grids. The complete grid was imaged by a tile scan of transmitted light at 5x magnification. The center of the grid (large green square), was imaged with transmitted light and fluorescence (red) by performing an autofocus tile scan at 20x magnification (pink FOV). Regions of interest were imaged by spinning disk confocal microscopy at 40x NA 0.9 (MIP small green squares).

Figure 2: Alignment of cryo-LM and cryo-SEM images utilizing MAPS software. After cryo-LM, the grid from Figure 1 was transferred to the cryo-SEM. A tile scan overview is taken by the SEM at 1 kV. Each tile is recorded with 3k x 2k pixels, 163 nm pixel size and 10 μs pixel dwell time. For alignment, first the 20x overview (blue area) is aligned to the SEM image based on the grid pattern. Subsequently, the MIPs from the 40x confocal recordings were aligned to the 20x overview based on the red fluorescent signal (small green areas).

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EMC Abstracts - https://emc-proceedings.com/abstract/mycobacterial-protein-weaponry-studied-by-cryo-correlative-microscopy/