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3 Janelia Publications

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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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