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62 Publications

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    01/01/11 | High resolution segmentation of neuronal tissues from low depth-resolution EM imagery.
    Glasner D, Hu T, Nunez-Iglesias J, Scheffer L, Xu C, Hess H, Fetter R, Chklovskii D, Basri R
    8th International Conference of Energy Minimization Methods in Computer Vision and Pattern Recognition Energy Minimization Methods in Computer Vision and Pattern Recognition. 2011;6819:261-72

    The challenge of recovering the topology of massive neuronal circuits can potentially be met by high throughput Electron Microscopy (EM) imagery. Segmenting a 3-dimensional stack of EM images into the individual neurons is difficult, due to the low depth-resolution in existing high-throughput EM technology, such as serial section Transmission EM (ssTEM). In this paper we propose methods for detecting the high resolution locations of membranes from low depth-resolution images. We approach this problem using both a method that learns a discriminative, over-complete dictionary and a kernel SVM. We test this approach on tomographic sections produced in simulations from high resolution Focused Ion Beam (FIB) images and on low depth-resolution images acquired with ssTEM and evaluate our results by comparing it to manual labeling of this data.

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    11/25/10 | Nanoscale architecture of integrin-based cell adhesions.
    Kanchanawong P, Shtengel G, Pasapera AM, Ramko EB, Davidson MW, Hess HF, Waterman CM
    Nature. 2010 Nov 25;468(7323):580-4. doi: 10.1038/nature09621

    Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity, and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signaling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a \~{}40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signaling layer containing integrin cytoplasmic tails, focal adhesion kinase, and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin’s polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.

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    01/01/10 | Increasing depth resolution of electron microscopy of neural circuits using sparse tomographic reconstruction.
    Veeraraghavan A, Genkin AV, Vitaladevuni S, Scheffer L, Xu C, Hess H, Fetter R, Cantoni M, Knott G, Chklovskii DB
    Computer Vision and Pattern Recognition (CVPR). 2010:1767-74. doi: 10.1109/CVPR.2010.5539846
    07/26/09 | Fast throughput low voltage scanning transmission electron microscope imaging of nano-resolution three dimensional tissue.
    Bolorizadeh M, Hess HF
    Microscopy and Microanalysis. 2009 Jul 26;15:642-3. doi: 10.1017/S1431927609092435
    Hess LabFetter Lab
    03/03/09 | Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.
    Shtengel G, Galbraith JA, Galbraith CG, Lippincott-Schwartz J, Gillette JM, Manley S, Sougrat R, Waterman CM, Kanchanawong P, Davidson MW, Fetter RD, Hess HF
    Proceedings of the National Academy of Sciences of the United States of America. 2009 Mar 3;106:3125-30. doi: 10.1073/pnas.0813131106

    Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

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    03/19/08 | Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling.
    Knott G, Marchman H, Wall D, Lich B
    The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2008 Mar 19;28(12):2959-64. doi: 10.1523/JNEUROSCI.3189-07.2008
    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.

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    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.

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    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.

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    06/17/94 | Near-field spectroscopy of the quantum constituents of a luminescent system.
    Hess HF, Betzig E, Harris TD, Pfeiffer LN, West KW
    Science. 1994 Jun 17;264(5166):1740-5. doi: 10.1126/science.264.5166.1740

    Luminescent centers with sharp (<0.07 millielectron volt), spectrally distinct emission lines were imaged in a GaAs/AIGaAs quantum well by means of low-temperature near-field scanning optical microscopy. Temperature, magnetic field, and linewidth measurements establish that these centers arise from excitons laterally localized at interface fluctuations. For sufficiently narrow wells, virtually all emission originates from such centers. Near-field microscopy/spectroscopy provides a means to access energies and homogeneous line widths for the individual eigenstates of these centers, and thus opens a rich area of physics involving quantum resolved systems.

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