Single Molecule Imaging in Live Embryos Using Lattice Light-Sheet Microscopy
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.
Key wordsSingle molecule imaging Single molecule kinetics Lattice light-sheet microscopy Drosophila melanogaster Live embryo imaging Single molecule fluorescence Transcription factor dynamics Single particle tracking Selective plane illumination microscopy
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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