Sullivan Kelley D, Brown Edward B
Department of Physics and Astronomy, University of Rochester, USA.
J Vis Exp. 2010 Feb 26(36):1636. doi: 10.3791/1636.
Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).
多荧光漂白恢复技术是一种用于测量大分子扩散系数（或类似传输参数）的显微镜技术，可应用于体外和体内生物系统。多荧光漂白恢复技术的操作过程是，先用强激光脉冲对荧光样品中的感兴趣区域进行漂白，然后减弱光束强度，并监测来自感兴趣区域外的仍具荧光的分子扩散进来取代被漂白分子时的荧光情况。我们将通过使激光束穿过普克尔盒（激光调制器）并沿着光路穿过激光扫描盒和物镜到达样品来开始演示。为简单起见，我们将使用水性荧光染料样品。然后，我们将确定适合我们样品的实验参数，包括监测和漂白功率、漂白持续时间、像素宽度（用于光子计数）以及荧光恢复时间。接下来，我们将描述获取恢复曲线的过程，该过程可通过LabVIEW（美国国家仪器公司，得克萨斯州奥斯汀）在很大程度上实现自动化，以提高通量。最后，使用最小二乘拟合算法将恢复数据拟合到适当的数学模型来确定扩散系数，这可以使用诸如MATLAB（美国数学工作公司，马萨诸塞州纳蒂克）之类的软件轻松编程实现。
