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Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates

Nature Protocols volume 12, pages 429–438 (2017)Cite this article

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Abstract

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

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Figure 1: Schematic of pad preparation and imaging.
Figure 2: Experimental setup.
Figure 3: Typical results from SLIP.

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Acknowledgements

The authors thank members of the Huang lab, especially M. Anjur-Dietrich, M. Rajendram, and A. Aranda-Diaz, for insightful discussions and beta-testing the SLIP software. This work was supported by a Stanford Agilent Fellowship and a Stanford Interdisciplinary Graduate Fellowship (to H.S.), a Stanford Graduate Fellowship (to A.C.), a Siebel Scholars Graduate Fellowship (to T.K.L.), funding through a National Institutes of Health (NIH) Biotechnology Training Grant (to T.K.L.), NIH Director's New Innovator Award DP2OD006466 (to K.C.H.), and National Science Foundation CAREER Award MCB-1149328 (to K.C.H.).

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Author notes

  1. Handuo Shi and Alexandre Colavin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California, USA

    Handuo Shi, Timothy K Lee & Kerwyn Casey Huang

  2. Biophysics Program, Stanford University, Stanford, California, USA

    Alexandre Colavin & Kerwyn Casey Huang

  3. Program in Biomedical Informatics, Stanford University School of Medicine, Stanford, California, USA

    Timothy K Lee

  4. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Kerwyn Casey Huang

Authors
  1. Handuo Shi
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  2. Alexandre Colavin
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Contributions

H.S., A.C., T.K.L., and K.C.H. designed the research; H.S., A.C., and T.K.L. performed the research; H.S. and K.C.H. analyzed the data; and H.S. and K.C.H. wrote the manuscript.

Corresponding author

Correspondence to Kerwyn Casey Huang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Photographs of agar pad and sample preparation.

(a) Melted agar is poured onto the bottom surface of a Singer PlusPlate and spread evenly by gently tilting and shaking the plate. (b) A flat agar surface is generally achieved when the plate is left on the benchtop to solidify without disturbance. The agar layer is ~2 mm in thickness. (c, d) Bacterial cultures are transferred onto an agar pad using a 96-well replicator pin. (e) After the agar pad absorbs all the liquid from the cultures, a large glass cover slip is used to cover the agar surface. (f) Immersion oil is applied evenly onto the cover slip for oil-immersion objectives.

Supplementary Figure 2 Optimization of TTL setup.

(a) Imaging time for a 5x5 grid of images for one strain scales linearly with camera exposure time. Reducing exposure time can expedite image acquisition. (b) Total imaging time for a 5x5 grid is dependent on stage acceleration time. In our configuration, an acceleration time of 40-50 ms is optimal.

Supplementary Figure 3 Variation in mean cellular dimensions calculated from individual fields of view is small.

The coefficient of variation of mean cell width (a) and length (b) across the fields of view from one of the wells in the experiment shown in Fig. 3a was ~3%. Data points are mean ± standard deviation for n > 30, horizontal solid lines are the mean value for cells across all fields of view, and dashed lines are ±5% of the mean. Position #7 happened to have no cells and thus is not included in the plot.

Supplementary information

Supplementary Text and Figures (PDF 401 kb)

Supplementary Data

Source code for SLIP software. (ZIP 1596 kb)

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Shi, H., Colavin, A., Lee, T. et al. Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates. Nat Protoc 12, 429–438 (2017). https://doi.org/10.1038/nprot.2016.181

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