Live cell spinning disk confocal microscope with single molecule localization module

具有单分子定位模块的活细胞转盘共聚焦显微镜

• 批准号: 514497685
• 金额: --
• 依托单位: Ruprecht-Karls-Universität Heidelberg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2023
• 资助国家: 德国
• 起止时间: 2022-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/514497685?language=en
• 关键词: Live; cell; spinning; disk; confocal

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.

荧光光学显微镜可以遵循蛋白质复合物的亚细胞动力学，或者 - 如果信号足够明亮，甚至是单蛋白。由于光的衍射极限，无法解析比用光的波长大约一半的距离的信号。共聚焦显微镜有助于改善对比度并消除焦点信号，但不能克服衍射极限。在此提案中，我们希望将旋转磁盘显微镜作为一种高度平行的，因此，将共焦成像与超分辨率模态结合在一起，以实现具有最终敏感性和高临时分辨率的最佳共聚焦对比度，并在3D中以及近乎分子分辨率。这将允许回答有关蛋白质和RNA在细胞器或特定标记识别细胞的其他特定位点的定位的问题。它还将允许分析形成兴奋性突触的胶质母细胞瘤细胞与神经元之间的相互作用。我们无法确切预测何时发生形成，即，我们需要在很长的时间内使用高帧速率体积数据捕获。因此，我们要求一个旋转的磁盘共聚焦系统，该系统使我们能够收集许多框架而不会造成光毒性或其他疾病。此外，为了克服基于衍射的分辨率限制，我们要求单分子定位显微镜（SMLM）的方式在同一样品上执行超分辨率显微镜。这种方式将根据风暴，DNA斑点或棕榈的组合最多允许3个通道，并导致20 nm的XY分辨率。 3D信息将通过发射光路径中的球形镜头获得，并通过合并在不同Z位置上获得的超分辨率堆栈来覆盖几微米。虽然使用TIRF照明实现了最高的对比度，但我们也希望进入细胞，因此需要一种方法来自动降低激光的入射角，以使照明超过TIRF场。重要的是，多模式仪器的所有组件都需要由软件机动和控制。这样可以保存用户特定的配置，这是将系统放置在多用户成像设施中的先决条件。总而言之，所需的多模式显微镜设置将在两个主要方向上提高我们的实验能力。 1）旋转磁盘共聚焦显微镜对3D中亚细胞结构的敏感和快速现场成像。 2）随后研究固定细胞中单蛋白和蛋白质复合物在20 nm处的定位。重要的是，通过使用同一平台，我们可以在样本中再次找到XY点，并将实时成像与超分辨率显微镜数据相关联。