TIRF microscope for single-molecule FRET applications

在单分子水平上观察生物分子机器的作用已经彻底改变了对无数生物过程的理解。突出的例子是运动蛋白马达沿着微管的手拉手行走机制或ATP合酶的旋转。传统的荧光显微镜技术有可能揭示生物分子沿着线性聚合物（如细胞骨架成分或DNA）的大规模运动，而单分子荧光共振能量转移（smFRET）的应用进一步使得在低纳米尺度上检测单个大分子内的动态运动成为可能。在这里，我们请求资金购买全内反射（TIRF）显微镜，通过单分子显微镜方法研究控制真核生物基因组三维组织的分子组装动力学。参与该提案的实验室对动态结合染色质以改变其拓扑结构的蛋白质机器有共同的兴趣，并随之改变其在细胞增殖过程中的功能；包括远程启动子-增强子DNA接触的转录因子组装或酶促DNA环挤压的运动复合物。解剖这些分子机器的复杂机制需要一种方法，不仅具有时间和空间分辨率，以跟踪单个事件随时间的变化，而且还具有同时检测构象变化的能力。所要求的仪器将能够在毫秒时间尺度上解析用不同荧光团标记的单分子，并通过smFRET实现时间分辨的分子构象动态测量。EMCCD相机对smFRET实验和生物精细样品的发射敏感检测是由sCMOS相机补充的，当获得更高的光子产量（bbb20光子/像素）时，可以在相同的分辨率下获得四倍大的面积。电动TIRF模块允许在TIRF和单个激光线的高倾斜和层压光学片（HILO）照明之间切换，以微调成像深度。一个温控箱可以在包括人类细胞系在内的各种模型系统的温度下进行成像。

Observing the action of biomolecular machines at the single-molecule level has revolutionized the understanding of countless biological processes. Prominent examples are the hand-over-hand walking mechanism of kinesin motors along microtubules or the rotation of the ATP synthase. Whereas conventional fluorescence microscopy techniques have the potential to reveal large-scale movements of biomolecules along linear polymers such as cytoskeletal components or DNA, the application of single-molecule fluorescence resonance energy transfer (smFRET) has furthermore enabled the detection of dynamic movements within individual macromolecules at the low-nanometer scale.Here, we request funds for the acquisition of a total internal reflection (TIRF) microscope to study the dynamics of molecular assemblies that control the three-dimensional organization of eukaryotic genomes by single-molecule microscopy methods. The laboratories that contribute to this proposal share a central interest in protein machines that dynamically bind chromatin to alter its topology and, concomitantly, its function during cell proliferation; including transcription factor assemblies that account for long-range promoter-enhancer DNA contacts or motor complexes that enzymatically drive the extrusion of DNA loops. Dissecting the intricate mechanisms of these molecular machines requires a methodology not only with the temporal and spatial resolution to follow individual events over time, but also with the capability to simultaneously detect conformational changes.The requested instrument will be able to resolve single molecules labelled with different fluorophores at millisecond timescale and enable time-resolved, dynamic measurements of molecular conformations by smFRET. Sensitive detection of emission for smFRET experiments and biologically delicate samples with an EMCCD camera is complemented by an sCMOS camera that allows the acquisition of a fourfold larger area at the same resolution whenever higher photon yields (>20 photons/pixel) are achieved. A motorized TIRF module allows for switching between TIRF and highly inclined and laminated optical sheet (HILO) illumination of individual laser lines to fine-tune the depth of imaging. A temperature-controlled box enables imaging at temperatures relevant to diverse model systems, including human cell lines.