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Micropilot: automation of fluorescence microscopy–based imaging for systems biology

Abstract

Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.

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Figure 1: Schematic workflow of Micropilot.
Figure 2: Assays of SEC31 and H2B-tubulin HeLa cells.
Figure 3: Examples and measurements of automatic FRAP on CBX1-EGFP cells.

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Acknowledgements

This study was technically supported by the use of the European Molecular Biology Laboratory Advanced Light Microscopy Facility and Information Technology Service Unit. We acknowledge C. Chapuis for the CBX1-EGFP BAC cell line expressing H2B-mCherry. This project was funded by grants to J.E. and R.P. (within the MitoCheck, MitoSys and Systems Microscopy consortia) by the European Commission (LSHG-CT-2004-503464, FP7/2007-2013-241548 and FP7/2007-2013-258068), to R.P. by the Landesstiftung Baden-Württemberg in the framework of the research program RNS/RNAi and within the Nationales Genomforschungsnetz-Plus consortium IG-CSG (01GS0865). T.H.T. is financed by the Deutsche Forschungsgemeinschaft Graduiertenkolleg GRK118.

Author information

Authors and Affiliations

Authors

Contributions

C.C. developed the 'Micropilot' software and drafted the manuscript. A.W. developed the Visual Basic for Applications macro and performed and analyzed the automatic FRAP experiments. T.H.T. developed the feature selection, extended classification to multiple channels and acquired and analyzed the ERES images. F.V. performed the ERES experiments. J.B. performed and analyzed the spindle length experiments. F.S. and U.L. developed the computer-aided microscopy interface and set up software prototypes. A.E. developed the communication of μManager with Micropilot. T.W. helped with image processing and object feature design. R.P. supervised the project. J.E. supervised the project and revised the manuscript.

Corresponding authors

Correspondence to Rainer Pepperkok or Jan Ellenberg.

Ethics declarations

Competing interests

F.S and U.L. filed a patent application covering the CAM approach (Patent Cooperation Treaty/European Patent 2007/059351/US patent application 20100103253). F.S. is employed by Leica Microsystems.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 317 kb)

Supplementary Software

Micropilot software source code, documentation, microscope scripts and demonstration images. (ZIP 37024 kb)

Supplementary Video 1

Example video of H2B-tubulin HeLa cells negative control from prophase recognition on. (MOV 1397 kb)

Supplementary Video 2

Example video of FRAP on CBX1-EGFP interphase. (MOV 2493 kb)

Supplementary Video 3

Example video of FRAP on CBX1-EGFP early prophase (slow recovery). (MOV 3780 kb)

Supplementary Video 4

Example video of FRAP on CBX1-EGFP late prophase (fast recovery). (MOV 5121 kb)

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Conrad, C., Wünsche, A., Tan, T. et al. Micropilot: automation of fluorescence microscopy–based imaging for systems biology. Nat Methods 8, 246–249 (2011). https://doi.org/10.1038/nmeth.1558

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