Single-cell systems biology by super-resolution imaging and combinatorial labeling

Program in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.
Nature Methods (Impact Factor: 32.07). 06/2012; 9(7):743-8. DOI: 10.1038/nmeth.2069
Source: PubMed


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.

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    • "Measuring RNA transcripts in single cells is now done in multiple ways, and similar conclusions about variability are emerging from the higher sensitivity methods. For individual genes, single molecule RNA fluorescence in situ hybridization (SM-RNA FISH) is highly informative (Femino et al. 1998; Raj et al. 2008), and multiplexed versions now enable multiple genes to be measured in parallel (Lubeck and Cai 2012). In principle, an advantage of SM-RNA FISH is the ability to accurately count the absolute number of transcripts in a cell. "
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    • "Dynamic reporters in general, however, require non-trivial genome engineering, and at best are limited to a few spectrally distinct fluorophores . By contrast, measurements such as fluorescence in situ hybridization (FISH) and immuno-staining do not have these limitations but only provide static snap shots [10] [32]. High throughput technologies can simultaneously measure numerous biomarkers in large populations at a single-cell resolution [10, 37–43], resulting in a snap-shot of a high-dimensional phenotypic space. "
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    • "65 of these sites. In another work, STORM, combined with FISH, was demonstrated to measure mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells (Lubeck and Cai, 2012). "
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