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

我们将建立一个超分辨率光学显微镜，能够在液氮温度下以多种颜色对样品进行三维成像。结构照明显微镜（SIM）可以克服衍射极限，采用巧妙调制的照明系统。由此产生的3D图像将允许将细胞内的结构特征定位在~100nm内。我们使用在液态乙烷中快速冷冻的样品，将样品固定在玻璃冰层中，使分子相互作用保持在原始状态。虽然这种方法比化学固定有许多优点，但它也降低了荧光团的光敏性，使光漂白问题减少，并增加了标记分子的有效可检测性。然而，样品必须保持在水的再结晶温度（大约-140℃）以下。在冷冻样品中定位标签需要高度的精度，以便在聚焦离子束（FIB）中进行关联，并随后通过冷冻电子显微镜（cryoEM）对分子细节进行成像。一个厚的细胞，在某一深度含有感兴趣的特征，可以用FIB“研磨”，因此只保留一层薄薄的原始保存细胞的冷冻层。为了获得最佳的低温电镜分辨率，薄层厚度应在150nm左右。靶向细胞中正确的150nm层需要非常精确的相关信号，但这种相关方法可以提供感兴趣的蛋白质周围分子环境的惊人细节，甚至可以通过结合低温电子断层扫描和亚层析成像平均来获得高分辨率的原位大分子复杂结构。显微镜的设计将允许我们进行4色3D扫描，并实时重建它们，为我们提供多个同步信号，以增加细胞环境。这也使我们能够在时间密集的FIB-铣削步骤之前筛选样品的适用性。我们麦吉尔大学的5名研究人员将使用这种显微镜工作流程来观察细胞骨架的动态特征，包括肌动蛋白丝的组装、微管的形成和中心体的拆卸，以及导电微生物生物膜的电互连，以及非包膜病毒感染的分子机制。

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.