Correlative light and electron microscopy under cryogenic conditions

冷冻电子断层扫描（cryo-ET）是目前唯一的工具，产生三维图像的完整细胞在近天然状态，因此是一个强大的技术，揭示细胞结构在体内。它依赖于通过在液氮温度下立即冷冻生物样品来固定生物样品，从而保护细胞的微妙结构。然而，在细胞的复杂环境中识别物体可能具有挑战性。一种解决方案是将冷冻ET与光学显微镜（LM）相关联，其中感兴趣的细胞对象用荧光标签定位。不幸的是，样品在冷冻前的移动会妨碍精确的相关性。因此，在用LM成像之前冷冻样品可以克服这一挑战。 在这里，我们要求升级我们现有的基础设施，以允许在低温条件下使用LM（包括超分辨率）对生物样品进行成像，升级自动数据收集的软件，以及用于随后使用cryo-ET成像的样品制备和处理设备。新的设备和软件将成为蒙特利尔大学生物化学系和牙科学院（UdeM）结构生物学机构设施的一部分。UdeM和麦吉尔的主要结构生物学家是共同申请人，这一事实反映了收购这种仪器的必要性和紧迫性。因此，我们希望获得的低温能力将很容易为蒙特利尔的整个研究界所用。

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.