Single-molecule spectrometer with multi-parameter fluorescence detection

艾宾浩斯研究小组研究蛋白质如何在细胞中折叠，以及它们如何在空间（即哺乳动物细胞内的空间）和时间（即年龄依赖性变化）中保持其功能。该小组进一步研究了蛋白质聚集和相分离的疾病状态。因此，开发和利用新的技术，可以直接在体内评估生物分子的动力学和热力学性质。所要求的仪器将允许在纳秒到毫秒的时间尺度上测量试管和细胞中的蛋白质折叠。它将进一步允许获得蛋白质在其原生细胞环境中的完整稳定性曲线。这样的测量将允许分析不同的物理化学因素（如拥挤、限制、水合作用）或生物因素（如伴侣、细胞应力）如何在细胞健康和疾病条件下调节折叠动力学和热力学（包括对折叠的标准自由能的焓和熵贡献）。这一信息不能用任何其他可用的技术获得，对小组中当前和计划的研究项目的成功至关重要。具体来说，计划中的仪器包括一个单分子光谱仪，具有多参数荧光检测（如FCS， FCCS， FLCS, nsFCS, smFRET，各向异性，FLIM）和快速红外激光加热。需要用于脉冲交错激发的多激光器设置以及用于同时记录不同偏振的多个荧光团的多检测器设置。Walla研究小组将专门使用这种配置进行多蛋白复合物的两两交叉相关分析，并开发一种高阶多相关荧光显微镜的一般方案。为了在不同温度下进行活细胞实验，需要一个稳定的培养系统。

The Ebbinghaus research group investigates how proteins fold in the cell and how they retain their functionality in space (i.e. spatially inside mammalian cells) and time (i.e. age-dependent changes). The group further investigates disease states in which proteins aggregate and phase separate. Therefore, novel techniques are developed and utilized with which kinetic and thermodynamic properties of biomolecules can be assessed directly in vivo. The requested instrument will allow to measure protein folding on nanosecond to millisecond timescales in the test tube and the cell. It will further allow to access the full stability curve of a protein in its native cellular environment. Such measurements will allow to analyze how different physicochemical factors (such as crowding, confinement, hydration) or biological factors (such as chaperones, cell stresses) modulate folding kinetics and thermodynamics (including the enthalpic and entropic contributions to the standard free energy of folding) in cellular health and disease conditions. This information cannot be obtained with any other available technique and will be crucial to the success of the current and planned research projects in the group. Specifically, the planned instrument encompasses a single molecule spectrometer with multi-parameter fluorescence detection (e.g. FCS, FCCS, FLCS, nsFCS, smFRET, anisotropy, FLIM) and fast infrared laser heating. A multi-laser setup for pulsed interleaved excitation as well as a multi-detector setup for simultaneous recording of multiple fluorophores in different polarizations is required. The Walla research group will specifically use this configuration for pairwise cross-correlational analysis of multi-protein complexes and develop a general scheme for higher-order multi-correlation fluorescence microscopy. To perform live cell experiments at different temperatures a stable incubation system is required.