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2 Janelia Publications

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    07/30/22 | Characterization, Comparison, and Optimization of Lattice Light Sheets
    Gaoxiang Liu , Xiongtao Ruan , Daniel E. Milkie , Frederik Görlitz , Matthew Mueller , Wilmene Hercule , Alison Kililea , Eric Betzig , Srigokul Upadhyayula
    bioRxiv. 2022 Jul 30:. doi: 10.1101/2022.07.30.502108

    Lattice light sheet microscopy excels at the non-invasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here we undertake a comprehensive theoretical and experimental analysis of various forms of light sheet microscopy which both demonstrates and explains why lattice light sheets provide significant improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. Development of a new type of “harmonic balanced” lattice light sheet is shown to improve performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.

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    05/11/22 | Super-resolution microscopy reveals actomyosin dynamics in medioapical arrays.
    Moore RP, Fogerson SM, Tulu US, Yu JW, Cox AH, Sican MA, Li D, Legant WR, Weigel AV, Crawford JM, Betzig E, Kiehart DP
    Molecular Biology of the Cell. 2022 May 11:mbcE21110537. doi: 10.1091/mbc.E21-11-0537

    Arrays of actin filaments (F-actin) near the apical surface of epithelial cells (medioapical arrays) contribute to apical constriction and morphogenesis throughout phylogeny. Here, super-resolution approaches (grazing incidence structured illumination, GI-SIM and lattice light sheet, LLSM) microscopy resolve individual, fluorescently labeled F-actin and bipolar myosin filaments that drive amnioserosa cell shape changes during dorsal closure in . In expanded cells, F-actin and myosin form loose, apically domed meshworks at the plasma membrane. The arrays condense as cells contract, drawing the domes into the plane of the junctional belts. As condensation continues, individual filaments are no longer uniformly apparent. As cells expand, arrays of actomyosin are again resolved - some F-actin turnover likely occurs, but a large fraction of existing filaments rearrange. In morphologically isotropic cells, actin filaments are randomly oriented and during contraction, are drawn together but remain essentially randomly oriented. In anisotropic cells, largely parallel actin filaments are drawn closer to one another. Our images offer unparalleled resolution of F-actin in embryonic tissue show that medioapical arrays are tightly apposed to the plasma membrane, are continuous with meshworks of lamellar F-actin and thereby constitute modified cell cortex. In concert with other tagged array components, super-resolution imaging of live specimens will offer new understanding of cortical architecture and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

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