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89 Janelia Publications

Showing 71-80 of 89 results
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    03/15/11 | Subnuclear segregation of genes and core promoter factors in myogenesis. (With commentary)
    Yao J, Fetter RD, Hu P, Betzig E, Tjian R
    Genes & Development. 2011 Mar 15;25(6):569-80. doi: 10.1073/pnas.1100640108

    Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. Here we report a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD (plant homeodomain) finger appears to be required for the sequestration of TAF3 to the nuclear interior. We suggest that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation.

    Commentary: Jie Yao in Bob Tijan’s lab used a combination of confocal microscopy and dual label PALM in thin sections cut from resin-embedded cells to show that certain core transcription components and their target genes are spatially segregated in myoblasts, but not in differentiated myotubes, suggesting that such spatial segregation may play a role in guiding cellular differentiation.

     

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    01/01/11 | Probing tension and dynamics in actomyosin mediated cell shape change.
    Higgins CD, Tulu US, Gao L, Betzig E, Kiehart DP, Goldstein B
    Molecular Biology of the Cell. 2011;22:
    01/01/11 | Pupil-segmentation-based adaptive optics for microscopy.
    Ji N, Milkie DE, Betzig E
    Proceedings of SPIE. 2011;7931:79310I. doi: 10.1117/12.876398

    Inhomogeneous optical properties of biological samples make it difficult to obtain diffraction-limited resolution in depth. Correcting the sample-induced optical aberrations needs adaptive optics (AO). However, the direct wavefront-sensing approach commonly used in astronomy is not suitable for most biological samples due to their strong scattering of light. We developed an image-based AO approach that is insensitive to sample scattering. By comparing images of the sample taken with different segments of the pupil illuminated, local tilt in the wavefront is measured from image shift. The aberrated wavefront is then obtained either by measuring the local phase directly using interference or with phase reconstruction algorithms similar to those used in astronomical AO. We implemented this pupil-segmentation-based approach in a two-photon fluorescence microscope and demonstrated that diffraction-limited resolution can be recovered from nonbiological and biological samples.

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    07/15/10 | Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. (With commentary)
    Frost NA, Shroff H, Kong H, Betzig E, Blanpied TA
    Neuron. 2010 Jul 15;67(1):86-99. doi: 10.1016/j.neuron.2010.05.026

    Within dendritic spines, actin is presumed to anchor receptors in the postsynaptic density and play numerous roles regulating synaptic transmission. However, the submicron dimensions of spines have hindered examination of actin dynamics within them and prevented live-cell discrimination of perisynaptic actin filaments. Using photoactivated localization microscopy, we measured movement of individual actin molecules within living spines. Velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

    Commentary: A nice application of single particle tracking PALM (sptPALM), showing the flow of actin in the spines of live cultured neurons. Since 2008, the PALM in our lab has largely become a user facility, available to outside users as well as Janelians. Grad student Nick Frost in Tom Blanpied’s group at the U. of Maryland Med School visited on a number of occasions to use the PALM, with training and assistance from Hari.

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    02/01/10 | Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues.
    Ji N, Milkie DE, Betzig E
    Nature Methods. 2010 Feb;7:141-7. doi: 10.1038/nmeth.1411

    Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction-limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 microm.

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    02/01/10 | Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues. (With commentary)
    Ji N, Milkie DE, Betzig E
    Nature Methods. 2010 Feb;7:141-7. doi: 10.1038/nmeth.1411

    Biological specimens are rife with optical inhomogeneities that seriously degrade imaging performance under all but the most ideal conditions. Measuring and then correcting for these inhomogeneities is the province of adaptive optics. Here we introduce an approach to adaptive optics in microscopy wherein the rear pupil of an objective lens is segmented into subregions, and light is directed individually to each subregion to measure, by image shift, the deflection faced by each group of rays as they emerge from the objective and travel through the specimen toward the focus. Applying our method to two-photon microscopy, we could recover near-diffraction-limited performance from a variety of biological and nonbiological samples exhibiting aberrations large or small and smoothly varying or abruptly changing. In particular, results from fixed mouse cortical slices illustrate our ability to improve signal and resolution to depths of 400 microm.

    Commentary: Introduces a new, zonal approach to adaptive optics (AO) in microscopy suitable for highly inhomogeneous and/or scattering samples such as living tissue. The method is unique in its ability to handle large amplitude aberrations (>20 wavelengths), including spatially complex aberrations involving high order modes beyond the ability of most AO actuators to correct. As befitting a technique designed for in vivo fluorescence imaging, it is also photon efficient.
    Although used here in conjunction with two photon microscopy to demonstrate correction deep into scattering tissue, the same principle of pupil segmentation might be profitably adapted to other point-scanning or widefield methods. For example, plane illumination microscopy of multicellular specimens is often beset by substantial aberrations, and all far-field superresolution methods are exquisitely sensitive to aberrations.

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    07/26/09 | Three-dimensional super-resolution imaging of thick biological samples.
    Vaziri A, Tang J, Shroff H, Shank C
    Microscopy and Microanalysis. 2009 Jul 26;15:36-7. doi: 10.1017/S1431927609092368
    06/16/09 | Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. (With commentary)
    Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS, Betzig E, Liphardt J
    PLoS Biology. 2009 Jun 16;7(6):e1000137. doi: 10.1371/journal.pbio.1000137

    The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

    Commentary: Our goal as tool developers is to invent methods capable of uncovering new biological insights unobtainable by pre-existing technologies. A terrific example is given by this paper, where grad students Derek Greenfield and Ann McEvoy in Jan Liphardt’s group at Berkeley used our PALM to image the size and position distributions of chemotaxis proteins in E. Coli with unprecedented precision and sensitivity. Their analysis revealed that the cluster sizes follow a stretched exponential distribution, and the density of clusters is highest furthest away from the largest (e.g., polar) clusters. Both observations support a model for passive self-assembly rather than active cytoskeletal assembly of the chemotaxis network.

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    12/23/08 | Multilayer three-dimensional super resolution imaging of thick biological samples.
    Vaziri A, Tang J, Shroff H, Shank CV
    Proceedings of the National Academy of Sciences of the United States of America. 2008 Dec 23;105(51):20221-6. doi: 10.1073/pnas.0810636105

    Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 microm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of approximately 10 microm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.

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    12/01/08 | Advances in the speed and resolution of light microscopy.
    Ji N, Shroff H, Zhong H, Betzig E
    Current Opinion in Neurobiology. 2008 Dec;18(6):605-16. doi: 10.1016/j.conb.2009.03.009

    Neurobiological processes occur on spatiotemporal scales spanning many orders of magnitude. Greater understanding of these processes therefore demands improvements in the tools used in their study. Here we review recent efforts to enhance the speed and resolution of one such tool, fluorescence microscopy, with an eye toward its application to neurobiological problems. On the speed front, improvements in beam scanning technology, signal generation rates, and photodamage mediation are bringing us closer to the goal of real-time functional imaging of extended neural networks. With regard to resolution, emerging methods of adaptive optics may lead to diffraction-limited imaging or much deeper imaging in optically inhomogeneous tissues, and super-resolution techniques may prove a powerful adjunct to electron microscopic methods for nanometric neural circuit reconstruction.

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