Gamari Benjamin D, Zhang Dianwen, Buckman Richard E, Milas Peker, Denker John S, Chen Hui, Li Hongmin, Goldner Lori S
Department of Physics, University of Massachusetts, Amherst, Massachusetts 01002.
AT&T Laboratories Research (ret.), Florham Park NJ, 07932.
Am J Phys. 2014 Jul;82(7):708-722. doi: 10.1119/1.4869188.
Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors.
单分子敏感显微镜和光谱技术正在改变生物物理和材料科学实验室。诸如荧光相关光谱法（FCS）和单分子敏感荧光共振能量转移（FRET）等技术现在在研究实验室中已普遍可用，但在教学实验室中却很少见。我们描述了一种廉价的电子设备和开源软件，它们弥补了这一差距，使对生物物理感兴趣的本科生能够获得最先进的研究能力。我们讨论了与FCS相关的强度相关函数以及如何从光子到达时间来确定它。我们通过使用适用于本科教学实验室的FCS测量蛋白质的流体动力学半径来演示该系统。基于现场可编程门阵列（FPGA）的电子设备易于构建，也适用于更高级的测量，并描述了几种应用。该系统实现后，具有8纳秒的定时分辨率，可以控制多达四个激光源，并可以从多达四个光子计数探测器收集信息。
