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

用于相关显微镜的 Cryo-3D 超分辨率光学显微镜

• 批准号: RTI-2022-00350
• 负责人: Strauss, Mike
• 金额: $ 10.93万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742484
• 关键词: Cryo; 3D; super; resolution; light

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.

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3D成像。结构照明显微镜（SIM）可以通过采用巧妙调制的照明系统来克服衍射极限。由此产生的3D图像将允许细胞内的结构特征定位到约100 nm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，将分子相互作用保持在原始状态。虽然这种方法具有许多优于化学固定的优点，但它也降低了荧光团的光敏性，使光漂白不太成为问题，并增加了标记分子的有效检测能力。然而，样品必须保持在水的重结晶温度以下（大约-140 ℃）。在冷冻样品内定位标记的高度精确度是需要在聚焦离子束（FIB）中进行相关性，以及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，在一定的深度包含一个感兴趣的特征，可以用FIB“研磨”，这样只有一个薄的冷冻层的原始保存的细胞仍然存在。为了通过cryoEM获得最佳分辨率，薄层应该大约为150 nm厚。靶向细胞中正确的150 nm层需要非常精确的相关信号，但是这种相关方法可以提供感兴趣的蛋白质周围的分子环境的壮观细节，并且甚至可以通过结合冷冻电子断层扫描和亚断层图像平均来产生高分辨率的原位大分子复合物结构。显微镜的设计将允许我们进行四色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研磨步骤之前筛选样品的适用性。我们在麦吉尔大学的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中的动态特征，包括肌动蛋白丝组装，微管形成和中心体解体，以及导电微生物生物膜的电互连，以及无包膜病毒感染的分子机制。