Live cell spinning disk confocal microscope with single molecule localization module

荧光显微镜可以跟踪蛋白质复合物的亚细胞动力学，如果信号足够亮，甚至可以跟踪单个蛋白质。由于光的衍射极限，不可能分辨出间隔接近所用光波长约一半的信号。共聚焦显微镜有助于提高对比度和消除失焦信号，但不能克服衍射极限。在本提案中，我们希望使用旋转盘显微镜作为一种高度并行的、因此快速的共聚焦成像方式，结合超分辨率模式，以达到最佳的细胞内共聚焦对比度，具有极高的灵敏度和三维高时间分辨率以及接近分子分辨率。这将允许回答关于蛋白质和rna定位到细胞器或由特定标记识别的细胞的其他特定位置的问题。它还将允许分析胶质母细胞瘤细胞和形成兴奋性突触的神经元之间的相互作用。我们无法准确预测地层何时发生，也就是说，我们需要在很长一段时间内以高帧率捕获体积数据。因此，我们要求一个旋转盘共聚焦系统，使我们能够收集许多帧而不造成光毒性或其他干扰。此外，为了克服基于衍射的分辨率限制，我们需要一种单分子定位显微镜（SMLM）的模式来对相同的标本进行超分辨率显微镜。基于STORM， DNA-paint或PALM的组合，这种模式将允许多达3个通道，并产生20 nm的xy分辨率。3D信息将通过发射光路中的球面透镜获得，并且通过合并在不同z轴位置获得的超分辨率堆栈将覆盖几微米。虽然使用TIRF照明可以实现最高的对比度，但我们还希望进入电池几微米，因此需要一种自动减小激光入射角的方法，以允许超出TIRF场的照明。重要的是，多模态仪器的所有组件都需要由软件驱动和控制。这允许保存用户特定的配置，这是将系统放置到多用户成像设施中的先决条件，正如本文所建议的那样。总之，所要求的多模态显微镜设置将在两个主要方向上提高我们的实验能力。1)利用自旋盘共聚焦显微镜实现亚细胞结构的快速实时三维成像。2)后续研究单个蛋白和蛋白复合物在20 nm固定细胞中的定位。重要的是，通过使用相同的平台，我们可以再次找到样品中的xy点，并将实时成像与超分辨率显微镜数据相关联。

Fluorescence light microscopy allows to follow subcellular dynamics of protein complexes or – if the signal is bright enough - even of single proteins. Due to the diffraction limit of light, it is not possible to resolve signals that are spaced closer than approximately half the wavelength of the used light. Confocal microscopy helps to improve contrast and eliminate out of focus signal but does not overcome the diffraction limit. In this proposal, we want to use spinning disk microscopy as a highly parallelized and therefore fast way of confocal imaging combined with a super-resolution modality, to achieve the best confocal contrast inside cells with ultimate sensitivity and high temporal resolution in 3D as well as near molecular resolution. This will allow answering questions on localization of proteins and RNAs to organelles or other specific sites of the cell identified by particular markers. It will also allow analyzing the interaction between glioblastoma cells and neurons forming an excitatory synapse. We cannot predict exactly when formation occurs, i.e., we need to capture with a high frame rate volumetric data over an extended period of time. We therefore ask for a spinning disk confocal system which allows us to collect many frames without inflicting phototoxicity or other disturbances. In addition, and to overcome the diffraction-based limitation in resolution, we ask for a modality for single molecule localization microscopy (SMLM) to perform super-resolution microscopy on the same specimens. This modality will allow for up to 3 channels based on the combination of STORM, DNA-paint or PALM and results in an xy resolution of 20 nm. 3D information will be obtained by a spherical lens in the emission light path and several micrometer will be covered by merging the super-resolution stacks acquired at different z-positions. While highest contrast is achieved using TIRF illumination, we want to also several micrometer into the cell, and therefore need a way to automatically decrease the incidence angle of the laser to allow illumination beyond the TIRF field. Importantly, all components of the multimodal instrument need to be motorized and controlled by software. This allows to save user specific configurations, a prerequisite for placing the system into a multi-user imaging facility, as proposed here. In summary, the requested multimodal microscope setup will advance our experimental abilities in two main directions. 1) Sensitive and fast live imaging of subcellular structures in 3D with spinning disk confocal microscopy. 2) Subsequent study of localization of single proteins and protein complexes in fixed cells at 20 nm. Importantly, by using the same platform, we can find xy points in the sample again and correlate live imaging with super-resolution microscopy data.