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62 Publications

Showing 41-50 of 62 results
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    06/27/14 | Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits.
    Hayworth KJ, Morgan JL, Schalek R, Berger DR, Hildebrand DG, Lichtman JW
    Frontiers in Neural Circuits. 2014 Jun 27;8:68. doi: 10.3389/fncir.2014.00068

    The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly-the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments.

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    01/26/14 | Correlative super-resolution fluorescence and metal-replica transmission electron microscopy.
    Sochacki KA, Shtengel G, Van Engelenburg SB, Hess HF, Taraska JW
    Nature Methods. 2014 Jan 26;11(3):305-8. doi: 10.1038/nmeth.2816

    We combine super-resolution localization fluorescence microscopy with transmission electron microscopy of metal replicas to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. We validate robust correlation on the scale of 20 nm by imaging endogenous clathrin (in two and three dimensions) and apply the method to find the previously unknown three-dimensional position of the endocytic protein epsin on clathrin-coated structures at the plasma membrane.

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    01/16/14 | Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.
    Van Engelenburg SB, Shtengel G, Sengupta P, Waki K, Jarnik M, Ablan SD, Freed EO, Hess HF, Lippincott-Schwartz J
    Science. 2014 Jan 16;343(6171):653-6. doi: 10.1126/science.1247786

    The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.

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    01/01/14 | Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM.
    Shtengel G, Wang Y, Zhang Z, Goh WI, Hess HF, Kanchanawong P
    Methods in Cell Biology. 2014;123:273-94. doi: 10.1016/B978-0-12-420138-5.00015-X

    Many biomolecules in cells can be visualized with high sensitivity and specificity by fluorescence microscopy. However, the resolution of conventional light microscopy is limited by diffraction to ~200-250nm laterally and >500nm axially. Here, we describe superresolution methods based on single-molecule localization analysis of photoswitchable fluorophores (PALM: photoactivated localization microscopy) as well as our recent three-dimensional (3D) method (iPALM: interferometric PALM) that allows imaging with a resolution better than 20nm in all three dimensions. Considerations for their implementations, applications to multicolor imaging, and a recent development that extend the imaging depth of iPALM to ~750nm are discussed. As the spatial resolution of superresolution fluorescence microscopy converges with that of electron microscopy (EM), direct imaging of the same specimen using both approaches becomes feasible. This could be particularly useful for cross validation of experiments, and thus, we also describe recent methods that were developed for correlative superresolution fluorescence and EM.

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    10/25/13 | Correlative photoactivated localization and scanning electron microscopy.
    Kopek BG, Shtengel G, Grimm JB, Clayton DA, Hess HF
    PLoS One. 2013 Oct 25;8(10):e77209. doi: 10.1371/journal.pone.0077209

    The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

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    08/02/13 | Electron microscopy reconstruction of brain structure using sparse representations over learned dictionaries.
    Hu T, Nunez-Iglesias J, Vitaladevuni S, Scheffer L, Xu S, Bolorizadeh M, Hess H, Fetter R, Chklovskii D
    IEEE Transactions on Medical Imaging. 2013 Aug 2;32(12):2179-88. doi: 10.1109/TMI.2013.2276018

    A central problem in neuroscience is reconstructing neuronal circuits on the synapse level. Due to a wide range of scales in brain architecture such reconstruction requires imaging that is both high-resolution and high-throughput. Existing electron microscopy (EM) techniques possess required resolution in the lateral plane and either high-throughput or high depth resolution but not both. Here, we exploit recent advances in unsupervised learning and signal processing to obtain high depth-resolution EM images computationally without sacrificing throughput. First, we show that the brain tissue can be represented as a sparse linear combination of localized basis functions that are learned using high-resolution datasets. We then develop compressive sensing-inspired techniques that can reconstruct the brain tissue from very few (typically 5) tomographic views of each section. This enables tracing of neuronal processes and, hence, high throughput reconstruction of neural circuits on the level of individual synapses.

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    04/17/12 | Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes.
    Kopek BG, Shtengel G, Xu CS, Clayton DA, Hess HF
    Proceedings of the National Academy of Science of the United States of America. 2012 Apr 17;109(16):6136-41. doi: 10.1073/pnas.1121558109

    Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.

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    01/01/12 | Imaging the post-fusion release and capture of a vesicle membrane protein.
    Sochacki KA, Larson BT, Sengupta DC, Daniels MP, Shtengel G, Hess HF, Taraska JW
    Nature Communications. 2012;3:1154. doi: 10.1038/ncomms2158

    The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter’s capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

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    10/01/12 | Super-resolution using sparse representations over learned dictionaries: reconstruction of brain structure using electron microscopy.
    Hu T, Nunez-Iglesias J, Vitaladevuni S, Scheffer L, Xu S, Bolorizadeh M, Hess H, Fetter R, Chklovskii D
    arXiv.org . 2012 Oct:

    A central problem in neuroscience is reconstructing neuronal circuits on the synapse level. Due to a wide range of scales in brain architecture such reconstruction requires imaging that is both high-resolution and high-throughput. Existing electron microscopy (EM) techniques possess required resolution in the lateral plane and either high-throughput or high depth resolution but not both. Here, we exploit recent advances in unsupervised learning and signal processing to obtain high depth-resolution EM images computationally without sacrificing throughput. First, we show that the brain tissue can be represented as a sparse linear combination of localized basis functions that are learned using high-resolution datasets. We then develop compressive sensing-inspired techniques that can reconstruct the brain tissue from very few (typically 5) tomographic views of each section. This enables tracing of neuronal processes and, hence, high throughput reconstruction of neural circuits on the level of individual synapses.

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    12/01/11 | Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction.
    Brown TA, Tkachuk AN, Shtengel G, Kopek BG, Bogenhagen DF, Hess HF, Clayton DA
    Molecular and Cellular Biology. 2011 Dec;31:4994-5010. doi: 10.1128/MCB.05694-11

    A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown.

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