High-speed TIRF-SIM/SIM super-resolution microscope for live-cell imaging

用于活细胞成像的高速 TIRF-SIM/SIM 超分辨率显微镜

• 批准号: 496847469
• 金额: --
• 依托单位: Zentrum für Medizinische Biotechnologie (ZMB)
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/496847469?language=en
• 关键词: speed; TIRF; SIM; super; resolution

Our understanding of complex dynamic cellular processes has advanced dramatically in recent years due to the development of cutting-edge super-resolution microscopy techniques that overcome the resolution limit of conventional light microscopy. Structured illumination microscopy (SIM) has proved to be one of the most competitive approaches given its relatively simple setup and broad applicability due to the use of a wide range of classical fluorophores and standard sample preparation protocols. Furthermore, the significant increase in acquisition speed due to spatial light modulator (SLM) devices for pattern generation now facilitates the routine use of SIM for live-cell imaging and the precise spatio-temporal investigation of dynamic subcellular processes on the molecular level. The participating groups in this proposal have so far gained critical insight into molecular mechanisms that control key cellular processes with physiological and pathological relevance. In particular, we have successfully studied processes ranging from DNA damage repair, golgi stress and endo-lysosomal damage response to cell migration and adhesion. The requested SIM microscope system will facilitate dissecting these molecular mechanisms with significantly higher resolution in space and time. Furthermore, many dynamic structures that are critical for cellular behaviour, such as the cortical actin cytoskeleton and adhesion sites are localized near the plasma membrane. Thus, we will combine super resolution live-cell imaging with total internal reflection fluorescence microscopy (TIRF-SIM) to take advantage of the dramatically improved signal-to-noise ratio for studying the molecular processes in these cell regions. Multi-colour read-outs and high-speed live-cell imaging combined with flexible ultra-fast switching between different imaging modes (TIRF-SIM/SIM/TIRF) will help deciphering the spatio-temporal coordination of key molecules identified in our previous studies with potential regulatory factors.Beyond steady state measurements, we will also combine the different imaging modalities with acute drug treatments and light-based manipulation of related signal networks components with an integrated photostimulation device. Using the wide range of fluorescence read-out capabilities, we will be able to perform multiple cell response measurements upon perturbations including organelle morphodynamics, protein localization and cytoskeletal and adhesion reorganization. Such perturbation-response studies will greatly advance our understanding of causalities in the signal networks and help dissecting complex regulatory mechanisms. Together, the unique combination of system components in the requested microscope will facilitate the dissection of a multitude of cellular processes with very high spatio-temporal precision and thus decisively expand our understanding of underlying molecular mechanisms.

近年来，由于尖端的超分辨率显微镜技术的发展，克服了传统光学显微镜的分辨率限制，我们对复杂的动态细胞过程的理解已经取得了显着进展。结构照明显微镜（SIM）已被证明是最具竞争力的方法之一，由于其相对简单的设置和广泛的适用性，由于使用了广泛的经典荧光团和标准样品制备协议。此外，由于用于图案生成的空间光调制器（SLM）设备，采集速度的显著增加现在促进了SIM用于活细胞成像的常规使用和在分子水平上动态亚细胞过程的精确时空调查。到目前为止，该提案的参与小组已经获得了对控制具有生理和病理相关性的关键细胞过程的分子机制的重要见解。特别是，我们已经成功地研究了从DNA损伤修复，高尔基体应激和内溶酶体损伤反应到细胞迁移和粘附的过程。所要求的SIM显微镜系统将有助于解剖这些分子机制，在空间和时间上具有显着更高的分辨率。此外，许多对细胞行为至关重要的动态结构，如皮质肌动蛋白细胞骨架和粘附位点都位于质膜附近。因此，我们将结合联合收割机的超分辨率活细胞成像与全内反射荧光显微镜（TIRF-SIM），以利用显着提高的信号-噪声比的研究在这些细胞区域的分子过程。多色读出和高速活细胞成像，结合不同成像模式之间的灵活超快切换（TIRF-SIM/SIM/TIRF）将有助于破译我们先前研究中确定的具有潜在调节因子的关键分子的时空协调。我们还将联合收割机与急性药物治疗和光-利用集成的光刺激设备对相关信号网络组件进行基于操作的方法。使用广泛的荧光读出能力，我们将能够进行多个细胞响应测量扰动后，包括细胞器形态动力学，蛋白质定位和细胞骨架和粘附重组。这种扰动-反应研究将极大地推进我们对信号网络中因果关系的理解，并有助于剖析复杂的调控机制。总之，所需显微镜中系统组件的独特组合将有助于以非常高的时空精度解剖大量细胞过程，从而决定性地扩大我们对潜在分子机制的理解。