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

      Science (New York, N.Y.)
      American Association for the Advancement of Science (AAAS)

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          Abstract

          Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

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          Most cited references55

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          Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System

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            Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.

            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.
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              Super-Resolution Video Microscopy of Live Cells by Structured Illumination

              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.
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                Author and article information

                Journal
                25342811
                4336192
                10.1126/science.1257998

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