Cryo-3D-super-resolution light microscope for correlative microscopy

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We will build a super-resolution light microscope capable of imaging samples at liquid nitrogen- temperature in 3D with multiple colours. Structured illumination microscopes (SIM) can overcome the diffraction limit by employing a cleverly modulated illumination system. The resulting 3D images will allow structural features within cells to be localized to within ~100nm. We use samples rapidly frozen in liquid ethane to fix the samples in a layer of vitreous ice, preserving molecular interactions in a pristine state. While this method has numerous advantages over chemical fixation, it also decreases the photosensitivity of the fluorophores, making photobleaching less of a problem, and increasing the effective detectability of labeled molecules. However, the sample must remain below the recrystallisation temperature of water (roughly --140 C). A high degree of precision in localizing labels within frozen samples is required for correlation in a focused ion beam (FIB), and subsequent imaging of molecular details by cryo-electron microscopy (cryoEM). A thick cell, containing a feature of interest at some depth, can be "milled" with a FIB, so that only a thin frozen layer of the pristinely preserved cell remains. To obtain the best resolution by cryoEM, the thin layer should be roughly 150nm thick. Targeting the correct 150nm layer in a cell requires very accurate correlative signal, but this correlative approach can deliver spectacular detail of the molecular environment surrounding proteins of interest, and can even result in high resolution in situ macromolecular complex structures by combining cryo--electron tomography and subtomogram averaging. The microscope design will allow us to perform 4--colour 3D scans and reconstruct them in real-time to give us multiple simultaneous signals for increased cellular context. This also allows us to screen samples for suitability before the time-intensive FIB--milling step. Our group of 5 investigators at McGill University will use this microscopy workflow to look at dynamic features in the cytoskeleton, including actin filament assembly, microtubule formation, and centrosome disassembly, as well as the electrical interconnection of conductive microbial biofilms, and the molecular mechanism of non-enveloped virus infection.