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Single-cell quantification of molecules and rates using open-source microscope-based cytometry

Nature Methods volume 4, pages 175–181 (2007)Cite this article

Abstract

Microscope-based cytometry provides a powerful means to study cells in high throughput. Here we present a set of refined methods for making sensitive measurements of large numbers of individual Saccharomyces cerevisiae cells over time. The set consists of relatively simple 'wet' methods, microscope procedures, open-source software tools and statistical routines. This combination is very sensitive, allowing detection and measurement of fewer than 350 fluorescent protein molecules per living yeast cell. These methods enabled new protocols, including 'snapshot' protocols to calculate rates of maturation and degradation of molecular species, including a GFP derivative and a native mRNA, in unperturbed, exponentially growing yeast cells. Owing to their sensitivity, accuracy and ability to track changes in individual cells over time, these microscope methods may complement flow-cytometric measurements for studies of the quantitative physiology of cellular systems.

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Figure 1: Quantitative information extracted by Cell-ID.
Figure 2: Steady-state distributions of YFP and YFP-Ste5.
Figure 3: Total fluorescence validation by comparison of Cell-ID–based measurements with flow cytometry.
Figure 4: YFP and YFP-Adh1tail maturation and decay.

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Acknowledgements

We thank J. Newman for use of a Becton-Dickinson LSR2 flow cytometer, A. Arkin for supplying HL-60 cells, and P. Walter for use of a Zeiss LSM 510 confocal microscope. We also thank one of the reviewers for the idea of using Z-stack of confocal images to validate the volume measurements described in the Supplementary Note. Work was under the “Alpha Project” at the Center for Quantitative Genome function, a US National Institutes of Health Center of Excellence in Genomic Science. The Alpha Project is supported by grant P50 HG02370 to R.B. from the US National Human Genome Research Institute (NHGRI). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NHGRI.

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Author notes

  1. Andrew Gordon and Alejandro Colman-Lerner: These authors contributed equally to this work.

Authors and Affiliations

  1. The Molecular Sciences Institute, 2168 Shattuck Avenue, Berkeley, 94704, California, USA

    Andrew Gordon, Alejandro Colman-Lerner, Tina E Chin, Kirsten R Benjamin, Richard C Yu & Roger Brent

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  1. Andrew Gordon
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Contributions

A.G. wrote the relevant software and with A.C.-L., performed the data analysis and developed the microscopy methods. A.C.-L. carried out the wet-lab experiments. T.E.C. constructed the plasmids and yeast strains. K.R.B. quantified the YFP-Ste5 derivative by western blot. R.C.Y., A.C.-L. and A.G. calibrated the microscope-based fluorescence measurements. R.B. provided input into project design and interpretation of results. A.G., A.C.-L. and R.B. wrote the bulk of the paper.

Corresponding authors

Correspondence to Andrew Gordon, Alejandro Colman-Lerner or Roger Brent.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Examples of Cell-ID methodology.

Supplementary Fig. 2

YFP vs YFP-ADH1tail sequence.

Supplementary Table 1

Sample single cell measurements.

Supplementary Software

Cell-ID code and description.

Supplementary Note

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Gordon, A., Colman-Lerner, A., Chin, T. et al. Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat Methods 4, 175–181 (2007). https://doi.org/10.1038/nmeth1008

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