Single Molecule sensitive CLSM for FLIM FRET

用于 FLIM FRET 的单分子敏感 CLSM

• 批准号: 519922049
• 金额: --
• 依托单位: Ludwig-Maximilians-Universität München (LMU)
• 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/519922049?language=en
• 关键词: Single; Molecule; sensitive; CLSM; FLIM

Single-molecule fluorescence microscopy has become an established technique with the applicant helping to shape numerous methodological developments. In particular, multicolor experiments and experiments with fluorescence resonance energy transfer allow the visualization of complex interactions of biomolecular complexes and photonic structures. In this context, fluorescence lifetime plays a crucial role. It is an orthogonal parameter and intensity-independent, which means that as an intensive variable it can provide more precise and robust insights. The requested instrument is a Swiss army knife regarding single-molecule confocal techniques. It will allow the applicant’s laboratory and the entire community in Munich to perform sophisticated single-molecule investigations at high throughput. The applicant established fluorescence lifetime imaging in his PhD thesis and has used this method in numerous applications. In the last five years, single molecule measurements focusing on fluorescence lifetime were used in over 20 peer-reviewed publications. So far, the applicant’s laboratory has built its own confocal fluorescence microscopy equipment and has played a formative role in method development. Meanwhile, the dynamic development has been completed and products from commercial suppliers have caught up in functionality and flexibility. Further, commercial instruments have advantages in terms of engineering performance, automation, and system integration. Here, we apply for a confocal, laser-scanning microscope optimized for single molecule detection and for numerous single-molecule spectroscopic applications such as single molecule measurements in solutions or on surfaces, fluorescence resonance energy transfer (FRET) microscopy, fluorescence correlation spectroscopy (FCS), and especially fluorescence lifetime imaging (FLIM) based on time-correlated single photon counting. Furthermore, multicolor measurements should be possible with high throughput and high automation, so that single-molecule measurements can be performed routinely, not only by specialists and method developers. To achieve the same throughput as a widefield microscope, autofocus and autonomous detection routines for immobilized single molecules will be required.

单分子荧光显微镜已成为一种成熟的技术，申请人帮助形成了许多方法学的发展，特别是，多色实验和荧光共振能量转移实验允许生物分子复合物和光子结构的复杂相互作用的可视化。它是一个正交参数并且与强度无关，这意味着作为一个密集变量，它可以提供更精确和稳健的见解，这对于单分子共聚焦技术来说是一把瑞士军刀。这申请人的实验室和慕尼黑的整个社区以高通量进行复杂的单分子研究。申请人在其博士论文中建立了荧光寿命成像，并在过去五年中在许多应用中使用了这种方法，重点是荧光。迄今为止，申请人的实验室已经建立了自己的共焦荧光显微镜设备，并在方法开发中发挥了重要作用，同时，动态开发也已完成，商业供应商的产品也已上市。向上此外，商业仪器在工程性能、自动化和系统集成方面具有优势，在这里，我们申请了针对单分子检测和众多单分子光谱应用进行优化的共焦激光扫描显微镜。溶液或表面上的单分子测量、荧光共振能量转移（FRET）显微镜、荧光相关光谱（FCS），尤其是基于时间相关单光子计数的荧光寿命成像（FLIM）此外，多色测量应该是可能的。具有高通量和高自动化能力，因此单分子测量不仅可以由专家和方法开发人员进行常规测量，还需要固定化单分子的自动对焦和自主检测程序，以实现与宽视野显微镜相同的通量。