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55 Janelia Publications

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    10/14/15 | The 2015 super-resolution microscopy roadmap.
    Hell SW, Sahl SJ, Bates M, Zhuang X, Heintzmann R, Booth MJ, Bewersdorf J, Shtengel G, Hess HF, Tinnefeld P
    Journal of Physics D: Applied Physics. 2015 Oct 14;48:443001. doi: 10.1088/0022-3727/48/44/443001

    Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physicsaddresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact.

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    06/08/15 | Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions.
    Case LB, Baird MA, Shtengel G, Campbell SL, Hess HF, Davidson MW, Waterman CM
    Nature Cell Biology. 2015 Jun 8;17(7):880-92. doi: 10.1038/ncb3180

    Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.

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    04/07/15 | A multi-layered protein network stabilizes the Escherichia coli FtsZ-ring and modulates constriction dynamics.
    Buss J, Coltharp C, Shtengel G, Yang X, Hess H, Xiao J
    PLoS Genetics. 2015 Apr 07;11(4):e1005128. doi: 10.1371/journal.pgen.1005128

    The prokaryotic tubulin homolog, FtsZ, forms a ring-like structure (FtsZ-ring) at midcell. The FtsZ-ring establishes the division plane and enables the assembly of the macromolecular division machinery (divisome). Although many molecular components of the divisome have been identified and their interactions extensively characterized, the spatial organization of these proteins within the divisome is unclear. Consequently, the physical mechanisms that drive divisome assembly, maintenance, and constriction remain elusive. Here we applied single-molecule based superresolution imaging, combined with genetic and biophysical investigations, to reveal the spatial organization of cellular structures formed by four important divisome proteins in E. coli: FtsZ, ZapA, ZapB and MatP. We show that these interacting proteins are arranged into a multi-layered protein network extending from the cell membrane to the chromosome, each with unique structural and dynamic properties. Further, we find that this protein network stabilizes the FtsZ-ring, and unexpectedly, slows down cell constriction, suggesting a new, unrecognized role for this network in bacterial cell division. Our results provide new insight into the structure and function of the divisome, and highlight the importance of coordinated cell constriction and chromosome segregation.

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    03/01/15 | Fixation-resistant photoactivatable fluorescent proteins for CLEM.
    Paez-Segala MG, Sun MG, Shtengel G, Viswanathan S, Baird MA, Macklin JJ, Patel R, Allen JR, Howe ES, Piszczek G, Hess HF, Davidson MW, Wang Y, Looger LL
    Nature Methods. 2015 Mar;12(3):215-8. doi: 10.1038/nmeth.3225

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

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    Hess LabFetter LabFlyEM
    02/16/15 | Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics.
    Hayworth KJ, Xu CS, Lu Z, Knott GW, Fetter RD, Tapia JC, Lichtman JW, Hess HF
    Nature Methods. 2015 Feb 16;12(4):319-22. doi: 10.1038/nmeth.3292

    Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm × 10 nm × 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 μm thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then 'volume stitched' back together, producing a final three-dimensional data set suitable for connectome tracing.

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    11/06/14 | Anesthetized- and awake-patched whole-cell recordings in freely moving rats using UV-cured collar-based electrode stabilization.
    Lee D, Shtengel G, Osborne JE, Lee AK
    Nature Protocols. 2014 Nov 06;9(12):2784-95. doi: 10.1038/nprot.2014.190

    Intracellular recording allows precise measurement and manipulation of individual neurons, but it requires stable mechanical contact between the electrode and the cell membrane, and thus it has remained challenging to perform in behaving animals. Whole-cell recordings in freely moving animals can be obtained by rigidly fixing ('anchoring') the pipette electrode to the head; however, previous anchoring procedures were slow and often caused substantial pipette movement, resulting in loss of the recording or of recording quality. We describe a UV-transparent collar and UV-cured adhesive technique that rapidly (within 15 s) anchors pipettes in place with virtually no movement, thus substantially improving the reliability, yield and quality of freely moving whole-cell recordings. Recordings are first obtained from anesthetized or awake head-fixed rats. UV light cures the thin adhesive layers linking pipette to collar to head. Then, the animals are rapidly and smoothly released for recording during unrestrained behavior. The anesthetized-patched version can be completed in ∼4-7 h (excluding histology) and the awake-patched version requires ∼1-4 h per day for ∼2 weeks. These advances should greatly facilitate studies of neuronal integration and plasticity in identified cells during natural behaviors.

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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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