Ranjit Suman, Lanzano Luca, Gratton Enrico
Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, Irvine, California.
Department of Nanophysics, Italian Institute of Technology, Genoa, Italy; Department of Physics, University of Genoa, Genoa, Italy.
Biophys J. 2014 Dec 16;107(12):2775-2785. doi: 10.1016/j.bpj.2014.08.041.
Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created.
荧光蛋白在细胞内的扩散已通过基于涨落的技术（荧光相关光谱法（FCS）或光栅扫描图像相关光谱法）或粒子追踪进行了测量。然而，这些方法都无法让我们测量图像中每个像素处荧光粒子的扩散。在每个单独像素处使用传统的单点FCS进行测量会导致细胞持续长时间暴露于激光下，最终使样品漂白。为克服这一限制，我们开发了一种我们认为是新的扫描方法，可同时构建细胞的荧光图像。在这种改进的光栅扫描新方法中，在采集图像时，激光在移动到下一行之前会对每条单独的线进行多次扫描。这种过程持续进行，直到整个区域被扫描完。这与原始的光栅扫描图像相关光谱法不同，在原始方法中，数据是通过对每一帧扫描一次然后对图像进行多次扫描来获取的。此方法所需的数据采集总时间比在每个像素处进行传统FCS分析所需的时间短得多。然而，在单个像素处，采集到的强度时间序列较短；需要对相关函数进行非常规分析以提取有关扩散的信息。这些相关数据已使用相量法进行分析，相量法是一种最初为分析荧光寿命成像（FLIM）图像而开发的免拟合方法。使用这种方法进行分析可估算图像中每个像素处荧光物质的平均扩散系数，因此，可以创建细胞的详细扩散图谱。
