PDF(Pubmed)
Abstract：
Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.
摘要：
由于细胞运动或其他细胞内过程，在细胞中执行准确的荧光相关光谱(FCS)测量可能具有挑战性。在这方面,最近已经表明,在短时段(分段FCS)中分析FCS数据对于提高细胞内FCS测量的准确性是非常有用的。这里,我们证明了分段FCS可以在商用激光扫描显微镜(LSM)上进行，即使没有专用的FCS模块。我们展示了如何在LeicaSP8共聚焦显微镜上获取数据，然后使用MATLAB中的自定义软件进行导出和处理。软件执行数据的分割以提取平均ACF并测量特定亚细胞区域中的扩散系数。首先,我们测量不同大小的荧光团在溶液中的扩散，表明可以在商业LSM中获得高质量的ACF。接下来,我们通过测量GFP在HeLa细胞核中的扩散系数来验证该方法，利用强度的变化来区分核质和核仁。不出所料,相对于核质，测得的GFP在核仁中的扩散系数较慢。最后,我们将该方法应用于表达PARP1染色体的HeLa细胞，以测量PARP1在不同亚细胞区域的扩散系数。我们发现与核质相比，PARP1在核仁中的扩散较慢。
