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Single molecule imaging in live embryos using lattice light-sheet microscopy

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

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

24 Scopus citations

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages541-559
Number of pages19
DOIs
StatePublished - 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1814
ISSN (Print)1064-3745

Keywords

  • Drosophila melanogaster
  • Lattice light-sheet microscopy
  • Live embryo imaging
  • Selective plane illumination microscopy
  • Single molecule fluorescence
  • Single molecule imaging
  • Single molecule kinetics
  • Single particle tracking
  • Transcription factor dynamics

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