TIRF microscope for single-molecule FRET applications

在单分子水平上观察生物分子机器的活动，彻底改变了人们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部行走机制或三磷酸腺苷合成酶的旋转。传统的荧光显微镜技术有可能揭示生物分子沿着细胞骨架成分或DNA等线性聚合物的大规模运动，而单分子荧光共振能量转移(SmFRET)的应用进一步使我们能够在低纳米尺度上检测单个大分子内的动态运动。在这里，我们申请资金购买全内反射(TIRF)显微镜，用单分子显微镜方法研究控制真核基因组三维组织的分子组装的动力学。为这一提议做出贡献的实验室都对蛋白质机器有核心兴趣，蛋白质机器动态结合染色质以改变其拓扑结构，并随之改变其在细胞增殖过程中的功能；包括解释长距离启动子-增强子DNA接触的转录因子组件或酶促驱动DNA环挤出的发动机复合体。剖析这些分子机器的复杂机制需要一种方法，不仅要有时间和空间分辨率来跟踪单个事件随着时间的推移，还要有能力同时检测构象变化。所需的仪器将能够在毫秒级分辨标记不同荧光团的单个分子，并能够通过smFRET对分子构象进行时间分辨的动态测量。利用EMCCD相机对smFRET实验和生物精细样品的发射进行灵敏检测的同时，还配备了一台sCMOS相机，只要获得更高的光子产额(&gt；20光子/像素)，就可以在相同分辨率下获取四倍大的面积。电动TIRF模块允许在TIRF和单个激光线路的高倾斜和叠层光学片(HILO)照明之间切换，以微调成像深度。温度控制盒可以在与包括人体细胞系在内的各种模型系统相关的温度下进行成像。

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.