Yadav Rajeev, Senanayake Kasun B, Comstock Matthew J
Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2022;2478:141-240. doi: 10.1007/978-1-0716-2229-2_8.
We present an instrument that combines high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy. Biological macromolecules exhibit complex conformation and stoichiometry changes in coordination with their motion and activity. To further our understanding of the complex machinery of life, we need methods that can simultaneously probe more than one degree of freedom of single molecules and complexes. Fluorescence optical tweezers, or "fleezers," combine the capabilities of optical tweezers and single-molecule fluorescence microscopy into a single instrument. Here we present the latest generation of a high-resolution fleezers instrument integrated with multicolor fluorescence spectroscopy. The tweezers portion of the instrument can manipulate biological macromolecules with pN scale forces while measuring subnanometer distances. Simultaneous with tweezers measurements, the multicolor fluorescence capability allows the direct observation of multiple molecules or multiple degrees of freedom which allows, for example, the observation of multiple proteins simultaneously within a complex. The instrument incorporates three fluorescence excitation lasers, all sourced from a single-mode optical fiber allowing a reliable alignment scheme, that allows, for example, three independent fluorescent probes or fluorescence resonance energy transfer (FRET) measurements and also increases flexibility in the choice of fluorescent probes. To avoid photobleaching and improve tweezers stability, the instrument implements a timesharing (using a single trap laser to produce a pair of traps via rapid switching between two locations) and interlacing (turning the trapping beam off when the fluorescence excitation beams are on and vice versa) scheme using acousto-optic modulators (AOM) to rapidly and precisely modulate lasers. Our latest "random phase" trap AOM control method obliterates previous residual trap positioning and bead position measurement errors. Here we present the general design principles and detailed construction and testing protocols for the instrument.
我们展示了一种结合了高分辨率光镊和多色共聚焦荧光光谱技术的仪器。生物大分子在与其运动和活性相协调时会表现出复杂的构象和化学计量变化。为了进一步理解生命的复杂机制，我们需要能够同时探测单个分子和复合物多个自由度的方法。荧光光镊，即“fleezers”，将光镊和单分子荧光显微镜的功能整合到一台仪器中。在此，我们展示了集成有多色荧光光谱技术的新一代高分辨率fleezers仪器。该仪器的光镊部分能够以皮牛尺度的力操纵生物大分子，同时测量亚纳米级别的距离。在进行光镊测量的同时，多色荧光功能允许直接观察多个分子或多个自由度，例如，可以同时观察复合物中的多个蛋白质。该仪器包含三台荧光激发激光器，均由单模光纤提供光源，从而实现可靠的对准方案，例如允许进行三种独立的荧光探针或荧光共振能量转移（FRET）测量，同时也增加了荧光探针选择的灵活性。为了避免光漂白并提高光镊的稳定性，该仪器采用了分时（使用单个捕获激光器通过在两个位置之间快速切换来产生一对捕获阱）和交错（当荧光激发光束开启时关闭捕获光束，反之亦然）方案，利用声光调制器（AOM）快速精确地调制激光。我们最新的“随机相位”捕获AOM控制方法消除了先前残留的捕获阱定位和珠子位置测量误差。在此，我们展示了该仪器总的设计原理、详细的构造以及测试方案。
