Correlative light and electron microscopy under cryogenic conditions

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Cryo electron tomography (cryo-ET) is currently the only tool that produces three-dimensional images of intact cells in near-native state and is therefore a powerful technique for revealing cellular structures in vivo. It relies on fixing biological samples by instantly freezing them at liquid nitrogen temperatures thus preserving the delicate structures of the cell. Identifying objects within the complex environment of the cell, however, can be challenging. One solution is to correlate cryo-ET with light microscopy (LM) in which cellular objects of interest are localized with fluorescent tags. Unfortunately, sample movement prior to freezing can prevent precise correlation. Therefore, freezing of the sample prior to imaging with LM can overcome this challenge. Here we are requesting an upgrade to our already existing infrastructure to allow for the imaging of biological samples under cryogenic conditions with LM (including super-resolution), software upgrade for automated data collection, and equipment for sample preparation and handling for subsequent imaging with cryo-ET. The new equipment and software will be part of the CFI-funded institutional Facility for Structural Biology in the Biochemistry Department and the Faculty of Dentistry at the Université de Montréal (UdeM). The need and urgency for the acquisition of this instrumentation are reflected by the fact that leading structural biologists from UdeM and McGill are co-applicants. Therefore, the cryogenic capabilities we aim to acquire will be readily accessible to the entire research community in Montréal.