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

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.

A new class of disordered elements controls DNA replication through initiator self-assembly

“…Embryos were dechorionated in 100% bleach for 90 s and placed on the surface of a 5 mm diameter glass coverslip using a fine haired paintbrush. The coverslip surface was made adhesive by applying a small drop of homemade glue solution (prepared by dissolving a roll of double-sided scotch tape in heptane overnight) (Mir et al, 2018).…”

“…Imaging was performed using a custom built Lattice Light-Sheet Microscope as previously described (Chen et al, 2014; Mir et al, 2018; Mir et al, 2017). For all experiments a 40 beam square lattice pattern was used with a minimum and maximum excitation numerical aperture of 0.44 and 0.55 respectively.…”

Dynamic multifactor hubs interact transiently with sites of active transcription in Drosophila embryos

“…Second, Rayleigh scattering, scattering from particles of sizes less than the wavelength of the imaging light (the phenomenon responsible for blue skies and red sunsets), scales as ~1/λ 4 where λ is the imaging wavelength. Thus, using longer wavelengths results in fewer photons being scattered and thus more photons being absorbed, emitted and collected from single molecules (Mir et al, 2018).…”

Mechanisms regulating zygotic genome activation

“…Increasingly sensitive, fast, and precise imaging technologies, such as super-resolution microscopy and light-sheet microscopy, have enabled researchers to image living embryos as they develop, and this capability has led to the identification of new structures and processes 139,176,177 . Advanced imaging methods have also improved the resolution with which ZGA is visualized.…”