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.

荧光显微镜可以跟踪蛋白质复合体的亚细胞动力学，或者--如果信号足够亮--甚至单个蛋白质的动力学。由于光的衍射限制，不可能分辨间隔比所用光的波长的大约一半更近的信号。共焦显微镜有助于提高对比度和消除离焦信号，但不能克服衍射限制。在这个方案中，我们希望使用旋转圆盘显微镜作为一种高度并行的、因此快速的共焦成像方法，并结合超分辨率模式，在细胞内实现最佳的共焦对比度，同时具有最终的灵敏度和高的3D和近分子分辨率的时间分辨率。这将允许回答有关蛋白质和RNA定位到细胞器或由特定标记识别的细胞其他特定位置的问题。它还可以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。我们不能准确地预测何时形成，即，我们需要在较长的一段时间内以高帧速率捕获体积数据。因此，我们要求一种旋转圆盘共聚焦系统，使我们能够收集许多帧，而不会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，我们要求一种用于单分子定位显微镜(SMLM)的设备来对相同的样品执行超分辨率显微镜。这种模式将允许基于Storm、DNA-Paint或Palm的组合最多3个通道，并产生20 nm的XY分辨率。在发射光路上通过球面透镜获得三维信息，并将在不同z位置采集的超分辨率叠加合并在一起覆盖几个微米。在使用TIRF照明获得最高对比度的同时，我们还希望将激光照射到细胞内几微米，因此需要一种方法来自动减少激光的入射角，以允许超出TIRF场的照明。重要的是，多模式仪器的所有组件都需要由软件进行电动和控制。这允许保存特定于用户的配置，这是将系统放入此处建议的多用户成像设施的先决条件。总而言之，所要求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)旋转圆盘共聚焦显微镜对亚细胞结构进行快速、灵敏的三维实时成像。2)在20 nm处对单个蛋白质和蛋白质复合体在固定细胞中的定位进行了研究。重要的是，通过使用相同的平台，我们可以再次找到样本中的XY点，并将实时成像与超分辨率显微镜数据相关联。