CL-SEM for studies on cellular components and infection

In the last years, the combination of different imaging modalities, in particular fluorescence optical light microscopy (FLM) and electron microscopy (EM) techniques, became one of the main challenges. Correlative Light Electron Microscopy (CLEM) methods allow to bridge the resolution gap between ultrastrucural studies and dynamic, functional analysis in cellular systems. Recent advances in light microscopy, by the development of super resolution technique, permit to overcome the optical diffraction limit and to reach 20nm resolution. But these techniques are still limited to imaging only fluorescently tagged proteins and do not provide information on their structural organization with respect to the other cellular components, showing the interest to correlate this information with the EM ultrastructure. We propose a new approach to study cytoskeleton elements inside the cells, by correlative light-scanning electron microscopy (CL-SEM) which combines stochastic optical reconstruction microscopy (STORM) or Structured illumination microscopy (SIM) and Scanning electron microscopy (SEM) data. We studied two solutions to investigate cytoskeleton rearrangement [1]: The first approach, is based on a gridded sample support [1] while the second one is based on a commercial sample holder (C.Zeiss Microscopy, Shuttle and Find module), which is compatible with both light and electron imaging systems. Cells are grown on gridded or non-gridded glass supports, depending on the chosen coordinates transfert method beetwen the two imaging systems. Then, a controlled extraction of the cytoplasm with immuno-localisation of target proteins (actin or septin) is performed. We applied these methodologies to study cytoskeleton reorganization during Shigella flexneri infection. Septin filaments which are associated to actin networks are recruited to different bacteria during infection. With our “home made” CLSEM (based on gridded glass support), we studied septin rearrangement around bacteria and observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures (Figure.1) [2]. Based on our previous work on septin cages, we investigated actin organization by a combination of high resolution optical imaging (SIM & STORM) and SEM. Our main goal is to increase the accuracy of the overlay between data obtained from high resolution and from electron microscopy observations. To this aim, we propose a workflow which allows a precise relocalization of targeted protein between both imaging systems, using fluoronanogold probes.