Abstract
To fully describe gene expression dynamics requires the ability to quantitatively capture expression in individual cells over time. Automated systems for acquiring and analyzing real-time images are needed to obtain unbiased data across many samples and conditions. We developed a microfluidics device, the RootArray, in which 64 Arabidopsis thaliana seedlings can be grown and their roots imaged by confocal microscopy over several days without manual intervention. To achieve high throughput, we decoupled acquisition from analysis. In the acquisition phase, we obtain images at low resolution and segment to identify regions of interest. Coordinates are communicated to the microscope to record the regions of interest at high resolution. In the analysis phase, we reconstruct three-dimensional objects from stitched high-resolution images and extract quantitative measurements from a virtual medial section of the root. We tracked hundreds of roots to capture detailed expression patterns of 12 transgenic reporter lines under different conditions.
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Acknowledgements
We are grateful for bioinformatic assistance from M. Kovtun and V. Popov and for technical assistance from N. Lebeck, Y. Nishimura, A. Woods, K. Lewis, B. Wohlrab and S. Satbhai. We thank H. Tsukagoshi, R. Sozzani, C. Topp and J. Van Norman for valuable discussions and critical reading of the manuscript. We also acknowledge J. Harer and Y. Wang for their assistance in algorithm design and software development and Z. Iwinski of Carl Zeiss MicroImaging Inc. for developing an interface for the Multitime application. We are grateful for seed donations from N. Geldner at the University of Lausanne (pCASP1∷GFP, pCASP2∷GFP, pCASP4∷GFP) and P. Doerner at the University of Edinburgh (pCycB1;1∷CycB1;1-GFP). This work was supported by US National Science Foundation award DBI-0953184 to U.O. and National Institutes of Health award P50-GM081883 and US National Science Foundation award IOS-1021619 to P.N.B. and U.O.
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Contributions
W.B. designed and conducted the RootArray experiments; B.M., B.T.M., D.L.M. and U.O. conceived and implemented the computational methods; B.M., B.T.M., I.P.-M. and W.B. analyzed the data; P.N.B., R.W.T., J.J., S.J.K., G.K.F. and R.L.C. conceived and developed the RootArray; U.O. and P.N.B. participated in experimental designs; and W.B., B.T.M., B.M., U.O. and P.N.B. wrote the paper.
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G.K.F., J.J., P.N.B., R.L.C., R.W.T. and W.B. have a financial interest in and/or were/are consultants for GrassRoots Biotechnology, which owns the rights to the RootArray technology.
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Supplementary Text and Figures
Supplementary Figures 1–14, Supplementary Tables 1–5 and 7 and Supplementary Notes 1 and 2 (PDF 15696 kb)
Supplementary Table 6
Gene expression change. False discovery rates for change of gene expression in the data set. (XLS 114 kb)
Supplementary Table 8
Growth rate analyses. Samples for growth rate analysis. (XLS 212 kb)
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Busch, W., Moore, B., Martsberger, B. et al. A microfluidic device and computational platform for high-throughput live imaging of gene expression. Nat Methods 9, 1101–1106 (2012). https://doi.org/10.1038/nmeth.2185
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DOI: https://doi.org/10.1038/nmeth.2185
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