Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution

Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

Structured-illumination microscopy can double the resolution of the wide-field fluorescence microscope, but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed SIM that is capable of 100 nm resolution at frame rates up to 11 Hz for several hundred time frames. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila S2 cells in the total internal reflection (TIRF) mode.