Program in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.
Nat Methods. 2012 Jun 3;9(7):743-8. doi: 10.1038/nmeth.2069.
Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral overlap between fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using fluorescence in situ hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells is a natural approach to bring systems biology into single cells.
荧光显微镜是一种强大的定量工具，可用于探索单细胞中的调控网络。然而，由于荧光团之间的光谱重叠，可同时测量的分子种类数量受到限制。在这里，我们展示了一种简单但通用的策略，通过使用光学超分辨率显微镜（SRM）和组合标记，可大大提高单细胞中分子的多重检测能力。作为原理验证，我们使用荧光原位杂交（FISH）用独特的荧光团组合标记 mRNA，并通过 SRM 解析荧光团的序列和组合。我们在单个酿酒酵母细胞中同时测量了 32 个基因的 mRNA 水平。这些实验表明，单细胞的组合标记和超分辨率成像，是将系统生物学引入单细胞的一种自然方法。
