ISS ChronosBH Fluorescence Lifetime Spectrometer

描述（由申请人提供）：荧光光谱学对我们理解分子和细胞生物学做出了无数重要贡献。生物系统中的内在和外在荧光团探针的基本性质对（亚）纳米尺度上的局部双分子环境非常敏感。荧光团探针性质（例如激发态荧光寿命衰减、偏振各向异性和能量转移）的测量可以反映大分子（例如蛋白质、酶、RNA和DNA）的详细结构、构象、运动、结合和动力学相互作用。例如，探针的荧光寿命的微小变化揭示了在10 E以下的短距离处的局部环境中的过程的特征（例如，猝灭和介电贡献）和高达100 E的长距离（例如，福斯特共振能量转移或FRET）。这允许直接测定蛋白质-蛋白质结合、复合物形成、分子接触、邻近性和总体构象变化的重要参数。这些寿命测量需要具有皮秒分辨率的高灵敏度寿命荧光计，例如所要求的ISS ChronosBH荧光寿命光谱仪。这款自动化仪器结合了稳健、快速且经过验证的时间相关单光子计数（TCSPC）方法和新型白色光连续可调皮秒脉冲激发源，可覆盖整个可见光到近红外光谱。该仪器可以快速测量从40 ps到1us的多组分寿命（在磷光模式下更长），具有高保真度，来自小体积（30 uL）和弱荧光样品。动力学研究可以用停流装置进行。该工具使创新的新的研究，例如11个不同的项目，从9个主要用户。这些项目包括离子通道中的分子运动、重组酶DNA复合物中的构象变化、淀粉样蛋白、人胰岛素降解酶的催化调节以及用于开发新荧光探针的蛋白质的化学合成。这种需求是迫切的，因为在芝加哥大学甚至更大的芝加哥地区都没有这种能力的仪器。为了提高可用性和影响力，该仪器将被放置在生物物理动力学研究所的跨学科纳米生物学设施共享核心中。此外，它将被整合到研究生生物物理实验室课程，从而最大限度地提高其曝光率和对未来生物和生物医学研究人员的影响。

DESCRIPTION (provided by applicant): Fluorescence spectroscopy has made myriad essential contributions to our understanding of molecular and cellular biology. Fundamental properties of intrinsic and extrinsic fluorophores probes in biological systems are exquisitely sensitive to the local bimolecular environment on (sub)-nanometer length scales. Measurement of fluorophore probe properties such as excited state fluorescence lifetime decay, polarization anisotropy, and energy transfer can reflect detailed structural, conformational, motional, binding, and dynamical interactions of macromolecules such as proteins, enzymes, RNA, and DNA. For instance, small changes in the fluorescence lifetime of a probe reveal the characteristics of processes in the local environment at short distances under 10 E (e.g., quenching and dielectric contributions) and long distances up to 100 E (e.g., Forster resonance energy transfer or FRET). This allows direct determination of important parameters of protein-protein binding, complex formation, molecular contact, proximity and overall conformational changes. These lifetime measurements require a highly sensitive lifetime fluorometer with picosecond resolution, such as the requested ISS ChronosBH Fluorescence Lifetime Spectrometer. This automated instrument combines the robust, fast, and proven Time-Correlated Single Photon Counting (TCSPC) method with a novel white light continuously tunable picosecond pulse excitation source that covers the entire visible to near-IR spectrum. The instrument can quickly measure multi-component lifetimes from 40ps to 1us (and longer in phosphorescence mode) with high fidelity from small volume (30uL) and weakly fluorescent samples. Kinetics studies are possible with a stop-flow apparatus. The instrument enables innovative new research as exemplified by 11 different projects from 9 major users. These projects encompass areas as diverse as molecular motions in ion channels, conformational changes in recombinase DNA complexes, amyloid proteins, catalysis regulation of human insulin degrading enzyme, and chemical synthesis of proteins for the development of new fluorescent probes. The need is urgent as there is no instrument of this capability at the University of Chicago or even the greater Chicago area. To increase availability and impact, the instrument will be placed in the interdisciplinary NanoBiology Facility shared core in the Institute for Biophysical Dynamics. Furthermore, it will be integrated in Graduate Biophysics lab curricula, thereby maximizing its exposure and impact on future biological and biomedical researchers.