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


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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Figure 1: Spatial ordering of fluorophores on mRNAs can be resolved by Gaussian centroid localization.
Figure 2: Super-resolution imaging enables combinatorial labeling of individual transcripts.
Figure 3: Validation of mRNA quantitation by super-resolution barcoding.
Figure 4: Single-cell expression profiles of 32 mRNAs.
Figure 5: Msn2 and Crz1 combinatorially affect target regulons.


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We credit B. Wold with discussions that led to this work. We thank M. Elowitz for lending space and equipment in his laboratory, T. Zhiyentayev, H.Q. Li and X. Wang for assistance with experiments, A. Raj for technical assistance with FISH, X.W. Zhuang and her group for STORM, and A. Eldar, S. Fraser, G.W. Li, J. Levine and J. Locke for discussion and reading of the manuscript. This work was supported by a Beckman Institute seed grant and a US National Institutes of Health New Innovator Award 1DP2OD008530.

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E.L. and L.C. performed the experiments, carried out the analysis and wrote the manuscript. L.C. conceived the idea and designed the experiments.

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Correspondence to Long Cai.

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The authors declare no competing financial interests.

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Supplementary Figures 1–21, Supplementary Tables 1–4, Supplementary Note (PDF 4012 kb)

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Lubeck, E., Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat Methods 9, 743–748 (2012).

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