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 intend to fluorescently label protein complexes, with the purpose of facilitating their segmentation in transmission electron tomograms. Our imaging strategy is to perform cryogenic selected volume tomography, keeping the specimen in a continuous frozen hydrated state. To this end, we are developing a cryogenic workflow in collaboration with FEI™, consisting of three microscopes: CorrSight™ light microscope, Scios Dualbeam™ scanning electron microscope with a focused ion beam and Tecnai Arctica™ cryo–transmission electron microscope. The focus of my contribution is on the integration of the CorrSight light microscope into the workflow. This translates to performing cryogenic light microscopy and correlating the images with EM for guided ion beam milling and tomography. The use of FM for guidance in EM is hindered by the large diffraction-limited resolution of light microscopy. Therefore, we will modify the CorrSight to perform cryogenic super-resolution imaging, using photoswitching fluorescent proteins as labels.
The specimens we will be investigating are professional antigen-presenting cells, infected by Mycobacterium spp.. These bacteria can resist phagosomal degradation and some species even translocate to the cytosol. An important mediator of mycobacterial virulence is the ESX-1 machinery, one of the five mycobacterial Type 7 Secretion Systems (T7SS). ESX-1 is capable of secreting effector proteins and is required for cytosolic translocation. However, it is not exactly known in which way this protein complex facilitates phagosomal escape. Structural information on T7SS assembly and conformations will help us to understand its function. To achieve this, subunits of the T7SS complex will be fused with fluorescent proteins and studied using our cryogenic selected volume tomography workflow.