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

用于软材料和生物材料的短时扩散和微流变学研究的荧光相关光谱 (FCS) 设置

• 批准号: RTI-2022-00700
• 负责人: Yethiraj, Anand
• 金额: $ 2.23万
• 依托单位: Memorial University of Newfoundland
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742987
• 关键词: Fluorescence; correlation; spectroscopy; FCS; setup

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).

软材料具有一些共同的特征。首先，它们在跨纳米级和显微镜的长度尺度上表现出自组装。其次，它们很容易被外部刺激力，食物从平衡中驱逐出来，并且可以实现不同的非平衡稳态作为能量注入的函数。第三，流体动力相互作用（其中大分子运动会影响周围溶剂的流动，这反过来影响其他大分子的运动）通常非常重要。为了量化流体动力相互作用（HI）的作用，能够跟踪短时标准的运动至关重要，在此期间，分子间碰撞很少见，在分子已经充分体验到环境以及所有中间时间尺度的长时间内。在我的实验室中，我们使用良好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缓慢和快速动力学。作为该策略的一部分，我们使用脉冲场梯度NMR作为研究纳米级大分子系统（例如蛋白质，多糖和大分子复合物）的慢速动态（即长期扩散）的强大方法。但是，我们的测量能力存在差距：我们缺少用于研究纳米级巨型分子系统的快速动态（即短期和中间时间的自我 - 散热器）的短期窗口（微秒至毫秒）。 当前的建议是探测器的请求，硬件跨和自动相关器以及控制计算机，以便在内部进行荧光相关光谱（FCS）测量。我们有光学元件（镜头和过滤器），高质量的光学表以及稳定的488nm（蓝色），50 MW激光，中性密度过滤器和功率计，最重要的是，构建FCS系统的专业知识。我们缺乏的以及构建全面运行FCS系统所需的内容是两个检测器，即相关器硬件和一个控制计算机。 FCS系统将在纪念馆内的生物物理组中充满活力的合作，包括大分子分子运动，其中包括软凝结物理学家，物理化学家，生物化学家和模拟专家。该设备对于进步和扩大我的NSERC资助研究计划的能力是至关重要的，以取得重大进步，并进一步发展我的学员，以成为熟练和竞争力的高素质人员（HQP）。