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92 Janelia Publications

Showing 1-10 of 92 results
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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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    11/22/21 | De novo endocytic clathrin coats develop curvature at early stages of their formation.
    Willy NM, Ferguson JP, Akatay A, Huber S, Djakbarova U, Silahli S, Cakez C, Hasan F, Chang HC, Travesset A, Li S, Zandi R, Li D, Betzig E, Cocucci E, Kural C
    Development Cell. 2021 Nov 22;56(22):3146-3159.e5. doi: 10.1016/j.devcel.2021.10.019

    Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.

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    09/30/21 | Cellular bases of olfactory circuit assembly revealed by systematic time-lapse imaging.
    Li T, Fu T, Wong KK, Li H, Xie Q, Luginbuhl DJ, Wagner MJ, Betzig E, Luo L
    Cell. 2021 Sep 30;184(20):5107. doi: 10.1016/j.cell.2021.08.030

    Neural circuit assembly features simultaneous targeting of numerous neuronal processes from constituent neuron types, yet the dynamics is poorly understood. Here, we use the Drosophila olfactory circuit to investigate dynamic cellular processes by which olfactory receptor neurons (ORNs) target axons precisely to specific glomeruli in the ipsi- and contralateral antennal lobes. Time-lapse imaging of individual axons from 30 ORN types revealed a rich diversity in extension speed, innervation timing, and ipsilateral branch locations and identified that ipsilateral targeting occurs via stabilization of transient interstitial branches. Fast imaging using adaptive optics-corrected lattice light-sheet microscopy showed that upon approaching target, many ORN types exhibiting "exploring branches" consisted of parallel microtubule-based terminal branches emanating from an F-actin-rich hub. Antennal nerve ablations uncovered essential roles for bilateral axons in contralateral target selection and for ORN axons to facilitate dendritic refinement of postsynaptic partner neurons. Altogether, these observations provide cellular bases for wiring specificity establishment.

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    04/01/20 | 3D ATAC-PALM: super-resolution imaging of the accessible genome.
    Xie L, Dong P, Chen X, Hsieh TS, Banala S, De Marzio M, English BP, Qi Y, Jung SK, Kieffer-Kwon K, Legant WR, Hansen AS, Schulmann A, Casellas R, Zhang B, Betzig E, Lavis LD, Chang HY, Tjian R, Liu Z
    Nature Methods. 2020 Apr 01;17(4):430-6. doi: 10.1038/s41592-020-0775-2

    To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–fluorescence in situ hybridization (FISH), RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

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    01/01/20 | Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans.
    Bai X, Melesse M, Sorensen Turpin CG, Sloan D, Chen C, Wang W, Lee P, Simmons JR, Nebenfuehr B, Mitchell D, Klebanow LR, Mattson N, Betzig E, Chen B, Cheerambathur D, Bembenek JN
    Development. 2020 Jan;147(1):1-16. doi: 10.1242/dev.181099

    While cytokinesis has been intensely studied, the way it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant embryonic divisions and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we found several unexpected alterations to cytokinesis including apical midbody migration in polarizing epithelial cells of the gut, pharynx and sensory neurons. Aurora B kinase, which is essential for several aspects of cytokinesis, remains apically localized in each of these tissues after internalization of midbody ring components. Aurora B inactivation disrupts cytokinesis and causes defects in apical structures, even if inactivated post-mitotically. Therefore, cytokinesis is implemented in a specialized way during epithelial polarization and Aurora B has a new role in the formation of the apical surface.

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    01/17/20 | Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
    Hoffman DP, Shtengel G, Xu CS, Campbell KR, Freeman M, Wang L, Milkie DE, Pasolli HA, Iyer N, Bogovic JA, Stabley DR, Shirinifard A, Pang S, Peale D, Schaefer K, Pomp W, Chang C, Lippincott-Schwartz J, Kirchhausen T, Solecki DJ, Betzig E, Hess HF
    Science. 2020 Jan 17;367(6475):. doi: 10.1126/science.aaz5357

    Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.

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    01/11/20 | Tiled reconstruction improves structured illumination microscopy.
    Hoffman DP, Betzig E
    bioRxiv. 2020 Jan 11:. doi: 10.1101/2020.01.06.895318

    Structured illumination microscopy (SIM) is widely used for fast, long-term, live-cell super-resolution imaging. However, SIM images can contain substantial artifacts if the sample does not conform to the underlying assumptions of the reconstruction algorithm. Here we describe a simple, easy to implement, process that can be combined with any reconstruction algorithm to alleviate many common SIM reconstruction artifacts and briefly discuss possible extensions.

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    09/02/19 | Software for lattice light-sheet imaging of FRET biosensors, illustrated with a new Rap1 biosensor.
    O’Shaughnessy EC, Stone OJ, LaFosse PK, Azoitei ML, Tsygankov D, Heddleston JM, Legant WR, Wittchen ES, Burridge K, Elston TC, Betzig E, Chew T, Adalsteinsson D, Hahn KM
    The Journal of Cell Biology. 2019 Sep 2;218(9):3153-3160. doi: 10.1083/jcb.201903019

    Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.

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    07/01/19 | Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth.
    David AF, Roudot P, Legant WR, Betzig E, Danuser G, Gerlich DW
    Journal of Cell Biology. 2019 Jul 01;218(7):2150-68. doi: 10.1083/jcb.201805044

    Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole-derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

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