Laboratory for Fluorescence Dynamics, University of California Irvine, Irvine, CA 92697, USA.
Beckman Laser Institute and Medical Clinic, University of California Irvine, Irvine, CA 92697, USA.
Int J Mol Sci. 2022 Aug 30;23(17):9840. doi: 10.3390/ijms23179840.
Fluorescence correlation spectroscopy (FCS) is an extremely versatile tool that has been widely used to measure chemical reaction rates, protein binding, nanoparticle-protein interactions, and biomolecular dynamics in vitro and in vivo. As an inherently micro-sized approach, FCS is compatible with high-throughput screening applications, as demanded for drug design, but typically limited to nanomolar concentrations, which restricts possible applications. Here, we show how massively parallel camera-based detection with side illumination can extend the usable concentration range of FCS more than 100-fold to measure low affinity processes. Our line illumination (LIM) approach is robust, fast (1 s acquisition times), and does not require any reference measurements to characterize the observation volume size.
荧光相关光谱学（FCS）是一种极其通用的工具，已被广泛用于测量体外和体内的化学反应速率、蛋白质结合、纳米颗粒-蛋白质相互作用和生物分子动力学。作为一种固有的微尺寸方法，FCS与高通量筛选应用兼容，这是药物设计所需要的，但通常限于纳摩尔浓度，这限制了可能的应用。在这里，我们展示了如何通过基于相机的大规模并行侧照明检测将 FCS 的可用浓度范围扩展 100 多倍，以测量低亲和力过程。我们的线照明（LIM）方法是稳健的、快速的（采集时间为 1 秒），并且不需要任何参考测量来表征观察体积大小。
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