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Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy

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Nanoscale Imaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1814))

Abstract

In the past decade, live-cell single molecule imaging studies have provided unique insights on how DNA-binding molecules such as transcription factors explore the nuclear environment to search for and bind to their targets. However, due to technological limitations, single molecule experiments in living specimens have largely been limited to monolayer cell cultures. Lattice light-sheet microscopy overcomes these limitations and has now enabled single molecule imaging within thicker specimens such as embryos. Here we describe a general procedure to perform single molecule imaging in living Drosophila melanogaster embryos using lattice light-sheet microscopy. This protocol allows direct observation of both transcription factor diffusion and binding dynamics. Finally, we illustrate how this Drosophila protocol can be extended to other thick samples using single molecule imaging in live mouse embryos as an example.

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Acknowledgments

The authors thank the Betzig lab at HHMI Janelia Research Campus for designs and advice on setting up the LLSM. We thank all members of the Darzacq, Tjian, Garcia, and Eisen labs for reagents, suggestions, and useful discussions. This work was supported by the California Institute of Regenerative Medicine (CIRM) LA1-08013 and the National Institutes of Health (NIH) UO1-EB021236 & U54-DK107980 to X.D., by the Burroughs Wellcome Fund Career Award at the Scientific Interface, the Sloan Research Foundation, the Human Frontiers Science Program, the Searle Scholars Program, and the Shurl and Kay Curci Foundation to H.G., a Howard Hughes Medical Institute investigator award to M.E., NSF Graduate Research Fellowships A.R. D.H. is a Pew-Stewart Scholar for Cancer Research supported by the Pew Charitable Trusts, the Alexander and Margaret Stewart Trust, the Siebel Stem Cell Institute, and NIH R01-CA196884.

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Mustafa Mir, Michael Stadler, Astou Tangara, Anders S. Hansen, Dirk Hockemeyer, Michael B. Eisen, Hernan Garcia & Xavier Darzacq

  2. Biophysics Graduate Group, University of California, Berkeley, CA, USA

    Armando Reimer, Michael B. Eisen & Hernan Garcia

  3. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA

    Michael B. Eisen

  4. Department of Integrative Biology, University of California, Berkeley, CA, USA

    Michael B. Eisen

  5. Department of Physics, University of California, Berkeley, CA, USA

    Hernan Garcia

Authors
  1. Mustafa Mir
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  2. Armando Reimer
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  3. Michael Stadler
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  4. Astou Tangara
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  5. Anders S. Hansen
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  6. Dirk Hockemeyer
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  7. Michael B. Eisen
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  8. Hernan Garcia
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  9. Xavier Darzacq
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Corresponding author

Correspondence to Xavier Darzacq .

Editor information

Editors and Affiliations

  1. Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska, USA

    Yuri L. Lyubchenko

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Mir, M. et al. (2018). Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy. In: Lyubchenko, Y. (eds) Nanoscale Imaging. Methods in Molecular Biology, vol 1814. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8591-3_32

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  • DOI: https://doi.org/10.1007/978-1-4939-8591-3_32

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8590-6

  • Online ISBN: 978-1-4939-8591-3

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