High resolution Microscope for Multi-parameter Fluorescence Image Spectroscopy

Fluorescence spectroscopy and imaging are important biophysical techniques to study the structure and dynamics of fluorescently labeled biomolecules and bridge these with their function under in vitro conditions or in living cells. To this end, we rely on the unique capabilities of recently developed fluorescence-based spectroscopy and imaging techniques in combination with multi-parameter detection that give maximum information and selectivity together with spatial resolution. The proposed microscope consists of two closely connected modules: (1) a confocal laser scanning unit with an ultimate dynamic range in time (picoseconds to minutes), and (2) a total internal reflection unit for super-resolution microscopy. Distances between two dyes on the molecular scale between 2 and 15 nm can be resolved by Förster Resonance Energy Transfer (FRET) measurements. If the inter-dye distances exceed 15 nm, super-resolution microscopy with single-molecule localization and colocalization analysis is most appropriate. For closing the gaps in time and space in fluorescence spectroscopy and imaging, we want to perform performing multi-modal microscopy on the same sample. We will realize a seamless implementation of a combination of FRET spectroscopy and super-resolution microscopy. In this way, we achieve molecular resolution and map structural and dynamic features of the biomolecular assemblies over a wide range of length scales. For gaining the maximum of information from the fluorescence signal, we want to probe more degrees of freedom in biomolecular dynamics by FRET. While the same distance distributions are obtainable from three two-color FRET experiments, information about the correlation of distance changes and thus the coordination of molecular movements is only obtained using three-color FRET. This is possible since three-color FRET experiments contain information about the co-occurrence of distances for the individual FRET pairs. In the proposed setup, we want to study single immobilized biomolecules under in vitro conditions and to perform seamless super-resolution FRET microscopy of biomolecules in a cellular context. To characterize single-molecule reactions, we want to employ microfluidics to shift reversible equilibria and to trigger reactions of immobilized molecules by adding ligands or binding partner or by varying reaction conditions (buffer, ionic strength, pH). The confocal module will be used to register single-molecule fluorescence intensity traces of single immobilized molecules for several colors with an ultimate time resolution. In addition, we will employ super-resolution FRET microscopy to study processes in biomolecular systems such as (1) Monitoring the membrane translocation and chaperone-dependent folding of a lipase, (2) Mapping functional molecules of innate immune defence on different length scales and (3) Deciphering the cellular signal initiation determinants of the apoptosis signaling complex.

荧光光谱和成像是研究荧光标记的生物分子的结构和动力学的重要生物物理技术，并在体外条件下或活细胞中桥接它们的功能。为此，我们依靠最近开发的基于荧光的光谱和成像技术的独特功能以及多参数检测的结合，可提供最大的信息和选择性以及空间分辨率。所提出的显微镜由两个紧密连接的模块组成：（1）具有时间最终动态范围的共聚焦激光扫描单元（picseconds toiceConds to Minits），以及（2）超级分辨率显微镜的总内部反射单元。通过Förster共振能量转移（FRET）测量，可以解决2至15 nm之间两个分子尺度的染料之间的距离。如果染色间距离超过15 nm，则最合适的是具有单分子定位和共定位分析的超分辨率显微镜。为了缩小荧光光谱和成像中时间和空间的间隙，我们希望在同一样品上执行多模式显微镜。我们将实现FRET光谱和超分辨率显微镜组合的无缝实现。通过这种方式，我们实现了分子分辨率和映射生物分子组件的结构和动态特征，这是在宽范围的长度范围内。为了从荧光信号中获取最大信息，我们希望通过FRET探测生物分子动力学的自由度。虽然从三个两色FRET实验中获得相同的距离分布，但有关距离变化相关的信息，因此仅使用三色fret获得分子运动的协调。这是可能的，因为三色FRET实验包含有关单个FRET对的距离共发生的信息。在拟议的设置中，我们希望在体外条件下研究单个固定的生物分子，并在细胞环境中对生物分子进行无缝的超分辨率FRET显微镜。为了表征单分子反应，我们希望采用微流体来移动可逆性等效性，并通过添加配体或结合伴侣或通过变化的反应条件（缓冲液，离子强度，pH）来触发固定分子的反应。共聚焦模块将用于记录单个固定分子的单分子荧光强度痕迹，用于具有最终时间分辨率的几种颜色。 In addition, we will employ super-resolution FRET microscopy to study processes in biomolecular systems such as (1) Monitoring the membrane translocation and chaperone-dependent folding of a lipase, (2) Mapping functional molecules of innate immunodefence on different length scales and (3) Deciphering The cellular signal initiative determines of the apoptosis signaling complex.