TIRF microscope for single-molecule FRET applications

适用于单分子 FRET 应用的 TIRF 显微镜

• 批准号: 512024179
• 金额: --
• 依托单位: Julius-Maximilians-Universität Würzburg
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/512024179?language=en
• 关键词: TIRF; microscope; single; molecule; FRET

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.

在单分子水平上观察生物分子机器的作用，彻底改变了对无数生物过程的理解。最突出的例子是驱动蛋白马达沿着微管的手与手交替行走机制或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（如细胞骨架组分或DNA）的大规模运动，而单分子荧光共振能量转移（smFRET）的应用进一步使得能够在低纳米尺度下检测单个大分子内的动态运动。我们申请资金用于购买全内反射（TIRF）显微镜，以研究通过单分子显微镜方法控制真核基因组三维组织的分子组装的动力学。对这一提议做出贡献的实验室对动态结合染色质以改变其拓扑结构并伴随其在细胞增殖期间的功能的蛋白质机器有着共同的核心兴趣;包括解释长距离启动子-增强子DNA接触或酶促驱动DNA环挤出的马达复合物的转录因子组件。剖析这些分子机器的复杂机制，需要一种方法，不仅与时间和空间分辨率，以跟踪随着时间的推移，个别事件，但也有能力同时检测conformational changes.The所要求的仪器将能够解决单分子标记不同的荧光团在毫秒的时间尺度，并使时间分辨，动态测量的分子构象的smFRET。使用EMCCD相机对smFRET实验和生物敏感样品的发射进行灵敏检测，并通过sCMOS相机进行补充，该相机允许在实现更高光子产率（>20光子/像素）时以相同分辨率采集四倍大的区域。电动TIRF模块允许在TIRF和高度倾斜和层压光学片（HILO）之间切换，以微调成像深度。温度控制箱能够在与不同模型系统（包括人类细胞系）相关的温度下进行成像。