Measuring fast dynamics in solutions and cells with a laser scanning microscope
Measuring fast dynamics in solutions and cells with a laser scanning microscope
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DOI：
10.1529/biophysj.105.062836
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发表时间：
2005-08-01
影响因子：
3.4
通讯作者：
Gratton, E
中科院分区：
文献类型：
--
作者：
Digman, MA;Brown, CM;Gratton, E
单点荧光相关光谱（FCS）允许在微秒到毫秒的时间范围内测量快速扩散和动态过程。对于活细胞的测量，图像相关光谱（ICS）和时间ICS扩展FCS方法的扩散时间长达数秒至数分钟，同时提供空间分辨的动态信息。然而，由于帧采集速率，ICS限于非常慢的动态。在这里，我们开发了新的扩展ICS探测空间相关性在以前无法访问的时间窗口。我们表明，使用标准的激光共聚焦成像技术（光栅扫描模式），我们不仅可以达到单点FCS的时间尺度，但也有ICS在提供空间信息的优势。这种新的方法称为光栅图像相关光谱（RICS），在扫描过程中快速测量细胞内的许多焦点，提供相同的FCS浓度和动态信息以及关于沿着扫描路径的点之间的空间相关性的信息。我们利用隐藏的时间结构的扫描方法，其中相邻的像素是几微秒，从而准确地测量动态过程，如分子扩散的微秒到秒的时间尺度。结合模拟数据，我们表明，广泛的扩散系数和浓度可以通过RICS测量。我们使用RICS首次确定了在CHOK 1细胞中稳定表达的paxillin-EGFP的空间分辨扩散。这种新型的数据分析在生物学中具有广泛的应用，它为使用任何标准激光共聚焦显微镜测量细胞系统中的快速和慢速动态过程提供了强大的工具。
Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation between points along the scanning path. Longer time dynamics are recovered from the information in successive lines and frames. We exploit the hidden time structure of the scan method in which adjacent pixels are a few microseconds apart thereby accurately measuring dynamic processes such as molecular diffusion in the microseconds-to-seconds timescale. In conjunction with simulated data, we show that a wide range of diffusion coefficients and concentrations can be measured by RICS. We used RICS to determine for the first time spatially resolved diffusions of paxillin-EGFP stably expressed in CHOK1 cells. This new type of data analysis has a broad application in biology and it provides a powerful tool for measuring fast as well as slower dynamic processes in cellular systems using any standard laser confocal microscope.