High resolution Microscope for Multi-parameter Fluorescence Image Spectroscopy

荧光光谱和成像是重要的生物物理技术，用于研究荧光标记的生物分子的结构和动力学，并在体外条件下或活细胞中将它们与其功能联系起来。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能力，结合多参数检测，提供最大的信息和选择性以及空间分辨率。该显微镜由两个紧密相连的模块组成：(1)具有最终动态范围（皮秒到分钟）的共聚焦激光扫描单元，以及(2)用于超分辨率显微镜的全内反射单元。两种染料在分子尺度上的距离在2到15纳米之间可以通过Förster共振能量转移（FRET）测量来解决。如果染料间距离超过15 nm，则采用超分辨显微镜进行单分子定位和共定位分析。为了缩小荧光光谱和成像在时间和空间上的差距，我们想对同一样品进行多模态显微镜检查。我们将实现FRET光谱和超分辨率显微镜相结合的无缝实现。通过这种方式，我们实现了分子分辨率，并在广泛的长度尺度上绘制了生物分子组装的结构和动态特征。为了从荧光信号中获得最大的信息，我们想用FRET探测更多的生物分子动力学自由度。虽然从三个双色FRET实验中可以获得相同的距离分布，但只有使用三色FRET才能获得有关距离变化相关性的信息，从而获得分子运动的协调。这是可能的，因为三色FRET实验包含的信息，共同出现的距离为个别FRET对。在提出的设置中，我们希望在体外条件下研究单个固定化生物分子，并在细胞环境下对生物分子进行无缝超分辨率FRET显微镜观察。为了表征单分子反应，我们希望利用微流体来改变可逆平衡，并通过添加配体或结合伙伴或通过改变反应条件（缓冲液，离子强度，pH值）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几种颜色的单分子荧光强度痕迹，具有最终的时间分辨率。此外，我们将使用超分辨率FRET显微镜来研究生物分子系统中的过程，如：(1)监测脂酶的膜易位和伴侣依赖性折叠，(2)在不同长度尺度上绘制先天免疫防御的功能分子，以及(3)解读细胞凋亡信号复合物的细胞信号起始决定因素。

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.