Main Menu (Mobile)- Block

Main Menu - Block

custom | custom

Search Results

general_search_page-panel_pane_1 | views_panes

6 Janelia Publications

Showing 1-6 of 6 results
Your Criteria:
    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.

    View Publication Page
    01/18/19 | Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution.
    Gao R, Asano SM, Upadhyayula S, Pisarev I, Milkie DE, Liu T, Singh V, Graves AR, Huynh GH, Zhao Y, Bogovic JA, Colonell J, Ott CM, Zugates CT, Tappan S, Rodriguez A, Mosaliganti KR, Sheu S, Pasolli HA, et al
    Science (New York, N.Y.). 2019 Jan 18;363(6424):eaau8302. doi: 10.1126/science.aau8302

    Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

    View Publication Page
    10/28/16 | Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER.
    Nixon-Abell J, Obara CJ, Weigel AV, Li D, Legant WR, Xu C, Pasolli HA, Harvey K, Hess HF, Betzig E, Blackstone C, Lippincott-Schwartz J
    Science (New York, N.Y.). 2016 Oct 28;354(6311):433-46. doi: 10.1126/science.aaf3928

    The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle that plays crucial roles in numerous cellular functions. We used emerging superresolution imaging technologies to clarify the morphology and dynamics of the peripheral ER, which contacts and modulates most other intracellular organelles. Peripheral components of the ER have classically been described as comprising both tubules and flat sheets. We show that this system consists almost exclusively of tubules at varying densities, including structures that we term ER matrices. Conventional optical imaging technologies had led to misidentification of these structures as sheets because of the dense clustering of tubular junctions and a previously uncharacterized rapid form of ER motion. The existence of ER matrices explains previous confounding evidence that had indicated the occurrence of ER “sheet” proliferation after overexpression of tubular junction–forming proteins.

    View Publication Page
    02/01/08 | High-density mapping of single-molecule trajectories with photoactivated localization microscopy. (With commentary)
    Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, Betzig E, Lippincott-Schwartz J
    Nature Methods. 2008 Feb;5(2):155-7. doi: 10.1038/nmeth.1176

    We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

    Commentary: As a stepping stone to true live cell PALM (see above), our collaborator Jennifer Lippincott-Schwartz suggested using the sparse photoactivation principle of PALM to track the nanoscale motion of thousands of individual molecules within a single living cell. Termed single particle tracking PALM (sptPALM), Jennifer’s postdocs Suliana Manley and Jen Gillette used the method in our PALM rig to create spatially resolved maps of diffusion rates in the plasma membrane of live cells. sptPALM is a powerful tool to study the active cytoskeletal or passive diffusional transport of individual molecules with far more measurements per cell than is possible without sparse photoactivation.

    View Publication Page
    04/07/07 | Developing photo activated localization microscopy
    George H. Patterson , Eric Betzig , Jennifer Lippincott-Schwartz , Harald F. Hess
    4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro. 2007 Apr 15:. doi: 10.1109/isbi.2007.357008

    In conventional biological imaging, diffraction places a limit on the minimal xy distance at which two marked objects can be discerned. Consequently, resolution of target molecules within cells is typically coarser by two orders of magnitude than the molecular scale at which the proteins are spatially distributed. Photoactivated localization microscopy (PALM) optically resolves selected subsets of protect fluorescent probes within cells at mean separations of <25 nanometers. It involves serial photoactivation and subsequent photobleaching of numerous sparse subsets of photoactivated fluorescent protein molecules. Individual molecules are localized at near molecular resolution by determining their centers of fluorescent emission via a statistical fit of their point-spread-function. The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this paper, some of the limitations for PALM imaging under current experimental conditions are discussed.

    View Publication Page
    09/15/06 | Imaging intracellular fluorescent proteins at nanometer resolution. (With commentary)
    Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF
    Science. 2006 Sep 15;313:1642-5. doi: 10.1126/science.1127344

    We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method–termed photoactivated localization microscopy–to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

    Commentary: The original PALM paper by myself and my friend and co-inventor Harald Hess, spanning the before- and after-HHMI eras. Submitted and publicly presented months before other publications in the same year, the lessons of the paper remain widely misunderstood: 1) localization precision is not resolution; 2) the ability to resolve a few molecules by the Rayleigh criterion in a diffraction limited region (DLR) does not imply the ability to resolve structures of arbitrary complexity at the same scale; 3) true resolution well beyond the Abbe limit requires the ability to isolate and localize hundreds or thousands of molecules in one DLR; and 4) certain photoactivatable fluorescent proteins (PA-FPs) and caged dyes can be isolated and precisely localized at such densities; yielding true resolution down to  20 nm. The molecular densities we demonstrate (105 molecules/m2) are more than two orders of magnitude greater than in later papers that year (implying ten-fold better true resolution) – indeed, these papers demonstrate densities only comparable to earlier spectral or photobleaching based isolation methods. We validate our claims by correlative electron microscopy, and demonstrate the outstanding advantages of PA-FPs for superresolution microscopy: minimally perturbative sample preparation; high labeling densities; close binding to molecular targets; and zero non-specific background.

    View Publication Page