Nanoscopy and NIC@IIT, CHT Erzelli, Istituto Italiano di Tecnologia, Via Enrico Melen 83, Building B, 16152 Genoa, Italy.
DIFILAB, Department of Physics, University of Genoa, Via Dodecaneso 33, 16143 Genoa, Italy.
Sensors (Basel). 2021 Mar 12;21(6):2010. doi: 10.3390/s21062010.
Since the introduction of super-resolution microscopy, there has been growing interest in quantifying the nanoscale spatial distributions of fluorescent probes to better understand cellular processes and their interactions. One way to check if distributions are correlated or not is to perform colocalization analysis of multi-color acquisitions. Among all the possible methods available to study and quantify the colocalization between multicolor images, there is image cross-correlation spectroscopy (ICCS). The main advantage of ICCS, in comparison with other co-localization techniques, is that it does not require pre-segmentation of the sample into single objects. Here we show that the combination of structured illumination microscopy (SIM) with ICCS (SIM-ICCS) is a simple approach to quantify colocalization and measure nanoscale distances from multi-color SIM images. We validate the SIM-ICCS analysis on SIM images of optical nanorulers, DNA-origami-based model samples containing fluorophores of different colors at a distance of 80 nm. The SIM-ICCS analysis is compared with an object-based analysis performed on the same samples. Finally, we show that SIM-ICCS can be used to quantify the nanoscale spatial distribution of functional nuclear sites in fixed cells.
自超分辨率显微镜问世以来，人们越来越感兴趣于定量分析荧光探针的纳米级空间分布，以更好地了解细胞过程及其相互作用。检查分布是否相关的一种方法是对多色采集进行共定位分析。在研究和量化多色图像之间共定位的所有可能方法中，有一种方法是图像互相关光谱学 (ICCS)。与其他共定位技术相比，ICCS 的主要优势在于它不需要将样品预先分割成单个物体。在这里，我们展示了将结构光照明显微镜 (SIM) 与 ICCS（SIM-ICCS）相结合，是一种从多色 SIM 图像定量共定位和测量纳米级距离的简单方法。我们在距离为 80nm 的包含不同颜色荧光团的 DNA 折纸模型样品的光学纳米标尺的 SIM 图像上验证了 SIM-ICCS 分析。将 SIM-ICCS 分析与对同一样品进行的基于对象的分析进行了比较。最后，我们证明了 SIM-ICCS 可用于定量固定细胞中功能性核位点的纳米级空间分布。
