High resolution Microscope for Multi-parameter Fluorescence Image Spectroscopy

荧光光谱和成像是研究荧光标记生物分子的结构和动力学，并在体外条件下或在活细胞中将其与功能联系起来的重要生物物理技术。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，与多参数检测相结合，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)具有极限动态范围(皮秒到分钟)的共焦激光扫描单元；(2)用于超分辨率显微镜的全内反射单元。通过Förster共振能量转移(FRET)测量，可以分辨分子尺度上2到15 nm之间的两种染料之间的距离。如果染料间的距离超过15 nm，单分子定位和共定位分析的超分辨显微镜是最合适的。为了缩小荧光光谱和成像在时间和空间上的差距，我们希望对同一样品进行多模式显微镜观察。我们将实现FRET光谱和超分辨率显微镜的无缝结合。通过这种方式，我们实现了分子分辨率，并绘制了广泛长度范围内生物分子组件的结构和动态特征图。为了最大限度地从荧光信号中获取信息，我们希望通过FRET来探索生物分子动力学中的更多自由度。虽然从三个双色FRET实验中可以获得相同的距离分布，但关于距离变化的相关性以及分子运动的协调性的信息只能使用三种颜色的FRET获得。这是可能的，因为三色FRET实验包含关于各个FRET对的距离共同出现的信息。在建议的设置中，我们希望在体外条件下研究单个固定化生物分子，并在细胞环境中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，我们希望使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)来触发固定化分子的反应。共聚焦模块将用于记录几种颜色的单分子固定分子的单分子荧光强度痕迹，并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)监测脂肪酶的膜转位和依赖于伴侣的折叠，(2)在不同长度尺度上定位天然免疫防御的功能分子，以及(3)破译细胞凋亡信号复合体的信号起始决定因素。

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.