Fluorescence correlation spectroscopy (FCS) setup for short-time diffusion and microrheology studies in soft and biological materials

柔软和有生命的材料有几个共同的特点。首先，它们在跨越纳米尺度和微米尺度的长度尺度上表现出自组装。其次，它们很容易被外力、食物等外部刺激赶出平衡状态，并且作为能量注入的函数，它们可以达到不同的非平衡稳定状态。第三，流体动力学相互作用的作用（大分子运动影响周围溶剂的流动，进而影响其他大分子的运动）通常是非常重要的。为了量化水动力相互作用（HI）的作用，关键是能够在短时间尺度上跟踪运动，在短时间尺度上，分子间碰撞很少，在长时间尺度上，分子完全经历了环境，以及在所有中间时间尺度上。在我的实验室里，我们使用精心打磨的荧光共聚焦显微镜和高速光学成像技术来阐明结构，并研究大型微米尺度胶体悬浮液的慢速和快速动力学。作为该策略的一部分，我们使用脉冲场梯度核磁共振作为研究纳米级大分子系统（如蛋白质，多糖和大分子复合物）的慢动力学（即长时间扩散率）的有力方法。然而，我们的测量能力存在差距：我们缺少研究纳米级大分子系统的快速动力学（即短时间和中时间自扩散率）的短时间窗口（微秒到毫秒）。目前的建议是要求检测器，硬件交叉和自相关器和控制计算机，以便在室内进行荧光相关光谱（FCS）测量。我们拥有光学器件（透镜和滤光片），高质量的光学表，稳定的488nm（蓝色），50 mW激光器，中性密度滤光片和功率计，最重要的是，我们拥有构建FCS系统的专业知识。我们所缺乏的和我们所需要的构建一个完全可操作的FCS系统是两个探测器，相关器硬件和一台控制计算机。FCS系统将用于纪念生物物理小组内部的大分子运动研究，其中包括软凝聚态物理学家、物理化学家、生物化学家和模拟专家。这些设备对于推进和扩大我的nserc资助的研究项目的能力至关重要，以取得重大进展，并进一步发展我的学员，使他们成为有技能和有竞争力的高素质人才（HQP）。

Soft and living materials have a few common characteristics. First, they exhibit self-assembly on length scales spanning the nanoscale and the microscale. Second, they are easily driven out of equilibrium by external stimuli - forces, food - and they can achieve different non-equilibrium steady states as a function of the energy injection. Third, the role of hydrodynamic interactions (where macromolecular motions affect the flow of the surrounding solvent, which in turn affects the motions of other macromolecules) is often very important. In order to quantify the role of hydrodynamic interactions (HI), it is critical to be able to track motions on short timescales, during which inter-molecular collisions are rare, at long times where the molecule has fully experienced the environment, and at all intermediate timescales. In my laboratory, we use well-honed fluorescence confocal microscopy and high-speed optical imaging techniques to elucidate structures and study both slow and fast dynamics of large, micron-scale colloidal suspensions. As part of this strategy, we use pulsed-field gradient NMR as a powerful way to study slow dynamics (i.e. long-time diffusivities) of nanoscale macromolecular systems such as proteins, polysaccharides, and macromolecular complexes. There is, however, a gap in our measurement capabilities: we are missing the short-time window (microseconds to milliseconds) for studying the fast dynamics (i.e., short-time and intermediate-time self-diffusivities) of nanoscale macromolecular systems. The current proposal is a request for detectors, a hardware cross- and auto-correlator and a control computer in order to carry out fluorescence correlation spectroscopy (FCS) measurements in house. We have the optics (lenses and filters), a high-quality optics table, and a stable 488nm (blue), 50 mW laser, neutral density filters and power meter, and most importantly, the knowhow to construct the FCS system. What we are lacking and what we require to construct a fully operational FCS system are two detectors, the correlator hardware and a control computer. The FCS system will be utilized by a vibrant collaboration within the Biophysics group at Memorial in the study of macromolecular motions, that includes soft condensed matter physicists, physical chemists, biochemists, and simulation experts. This equipment will be essential for progressing and expanding the capacity of my NSERC-funded research programs to make major advances and further the development of my trainees to become skillful and competitive highly qualified personnel (HQP).