Advertisement

Fluorescence-Based High-Throughput and Targeted Image Acquisition and Analysis for Phenotypic Screening

  • Manuel GunkelEmail author
  • Jan Philipp Eberle
  • Holger Erfle
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)

Abstract

Applying the right acquisition method in a fluorescence imaging-based screening context is of great importance to obtain an appropriate readout and to select the right scale of the screen. In order to save imaging time and data, we have developed routines for multiscale targeted imaging, providing both a broad overview of a sample and additional in-depth information for targets of interest identified within the screen. These objects can be identified and acquired on-the-fly by an interconnection of image acquisition and image analysis.

Key words

High-throughput screening High content screening Targeted imaging Automated imaging KNIME 

Notes

Acknowledgment

The authors wish to thank Michael Berthold and the KNIME/KNIP developers for their help with the development of the KNIME nodes and workflows. This work was funded within the project CancerTelSys (grant number 01ZX1302) in the e:Med program, the project HD-HuB (grant number 031A537C) in the de.NBI program and within the project RNA-Code (grant number 031A298) in the e:Bio program, all of the German Federal Ministry of Education and Research (BMBF). The ViroQuant-CellNetworks RNAi Screening Facility was also supported by the CellNetworks-Cluster of Excellence (grant number EXC81).

References

  1. 1.
    Neumann B, Held M, Liebel U, Erfle H, Rogers P, Pepperkok R, Ellenberg J (2006) High-throughput RNAi screening by time-lapse imaging of live human cells. Nat Methods 3(5):385–390. doi: 10.1038/nmeth876 CrossRefPubMedGoogle Scholar
  2. 2.
    Neumann B, Walter T, Hériché J-K, Bulkescher J, Erfle H, Conrad C, Rogers P, Poser I, Held M, Liebel U, Cetin C, Sieckmann F, Pau G, Kabbe R, Wünsche A, Satagopam V, Schmitz MHA, Chapuis C, Gerlich DW, Schneider R, Eils R, Huber W, Peters J-M, Hyman AA, Durbin R, Pepperkok R, Ellenberg J (2010) Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464(7289):721–727. doi: 10.1038/nature08869 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Gunkel M, Flottmann B, Heilemann M, Reymann J, Erfle H (2014) Integrated and correlative high-throughput and super-resolution microscopy. Histochem Cell Biol 141(6):597–603. doi: 10.1007/s00418-014-1209-y CrossRefPubMedGoogle Scholar
  4. 4.
    Flottmann B, Gunkel M, Lisauskas T, Heilemann M, Starkuviene V, Reymann J, Erfle H (2013) Correlative light microscopy for high-content screening. Biotechniques 55(5):243–252. doi: 10.2144/000114099 CrossRefPubMedGoogle Scholar
  5. 5.
    Eliceiri KW, Berthold MR, Goldberg IG, Ibanez L, Manjunath BS, Martone ME, Murphy RF, Peng H, Plant AL, Roysam B, Stuurman N, Swedlow JR, Tomancak P, Carpenter AE (2012) Biological imaging software tools. Nat Methods 9(7):697–710. doi: 10.1038/nmeth.2084 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Conrad C, Wünsche A, Tan TH, Bulkescher J, Sieckmann F, Verissimo F, Edelstein A, Walter T, Liebel U, Pepperkok R, Ellenberg J (2011) Micropilot: automation of fluorescence microscopy-based imaging for systems biology. Nat Methods 8(3):246–249. doi: 10.1038/nmeth.1558 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tischer C, Hilsenstein V, Hanson K, Pepperkok R (2014) Adaptive fluorescence microscopy by online feedback image analysis. Methods Cell Biol 123:489–503. doi: 10.1016/B978-0-12-420138-5.00026-4 CrossRefPubMedGoogle Scholar
  8. 8.
    Osterwald S, Wörz S, Reymann J, Sieckmann F, Rohr K, Erfle H, Rippe K (2011) A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway. Biotechnol J. doi: 10.1002/biot.201000474 PubMedGoogle Scholar
  9. 9.
    Gunkel M, Beil N, Beneke J, Reymann J, Erfle H (2015) Fluorescence microscopy-based RNA interference screening. Methods Mol Biol 1251:59–66. doi: 10.1007/978-1-4939-2080-8_4 CrossRefPubMedGoogle Scholar
  10. 10.
    Erfle H, Neumann B, Liebel U, Rogers P, Held M, Walter T, Ellenberg J, Pepperkok R (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc 2(2):392–399. doi: 10.1038/nprot.2006.483 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Manuel Gunkel
    • 1
    Email author
  • Jan Philipp Eberle
    • 1
  • Holger Erfle
    • 1
  1. 1.BioQuant Center, ViroQuant-CellNetworks RNAi Screening FacilityUniversity of HeidelbergHeidelbergGermany

Personalised recommendations