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110 Publications

Showing 71-80 of 110 results
12/07/12 | Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.
Gao L, Shao L, Higgins CD, Poulton JS, Peifer M, Davidson MW, Wu X, Goldstein B, Betzig E
Cell. 2012 Dec 7;151(6):1370-85. doi: 10.1016/j.cell.2012.10.008

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we demonstrate substantially reduced photodamage when imaging rapid morphological changes in D. discoideum cells, as well as improved contrast and resolution at depth within developing C. elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

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11/01/12 | Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.
Giurumescu CA, Kang S, Planchon TA, Betzig E, Bloomekatz J, Yelon D, Cosman P, Chisholm AD
Development. 2012 Nov;139(22):4271-9. doi: 10.1242/dev.086256

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to  350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

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06/01/12 | Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras.
Shao L, Winoto L, Agard DA, Gustafsson MG, Sedat JW
Journal of Microscopy. 2012 Jun;246:229-36. doi: 10.1111/j.1365-2818.2012.03604.x

In an interferometer-based fluorescence microscope, a beam splitter is often used to combine two emission wavefronts interferometrically. There are two perpendicular paths along which the interference fringes can propagate and normally only one is used for imaging. However, the other path also contains useful information. Here we introduced a second camera to our interferometer-based three-dimensional structured-illumination microscope (I(5)S) to capture the fringes along the normally unused path, which are out of phase by π relative to the fringes along the other path. Based on this complementary phase relationship and the well-defined phase interrelationships among the I(5)S data components, we can deduce and then computationally eliminate the path length errors within the interferometer loop using the simultaneously recorded fringes along the two imaging paths. This self-correction capability can greatly relax the requirement for eliminating the path length differences before and maintaining that status during each imaging session, which are practically challenging tasks. Experimental data is shown to support the theory.

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03/09/12 | Triggering a cell shape change by exploiting preexisting actomyosin contractions.
Roh-Johnson M, Shemer G, Higgins CD, McClellan JH, Werts AD, Tulu US, Gao L, Betzig E, Kiehart DP, Goldstein B
Science. 2012 Mar 9;335(6073):1232-5. doi: 10.1126/science.1217869

Apical constriction changes cell shapes, driving critical morphogenetic events, including gastrulation in diverse organisms and neural tube closure in vertebrates. Apical constriction is thought to be triggered by contraction of apical actomyosin networks. We found that apical actomyosin contractions began before cell shape changes in both Caenorhabitis elegans and Drosophila. In C. elegans, actomyosin networks were initially dynamic, contracting and generating cortical tension without substantial shrinking of apical surfaces. Apical cell-cell contact zones and actomyosin only later moved increasingly in concert, with no detectable change in actomyosin dynamics or cortical tension. Thus, apical constriction appears to be triggered not by a change in cortical tension, but by dynamic linking of apical cell-cell contact zones to an already contractile apical cortex.

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Ji LabBetzig LabSvoboda Lab
01/03/12 | Characterization and adaptive optical correction of aberrations during in vivo imaging in the mouse cortex.
Ji N, Sato TR, Betzig E
Proceedings of the National Academy of Sciences of the United States of America. 2012 Jan 3;109:22-7. doi: 10.1073/pnas.1109202108

The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca(2+) imaging in single neurons.

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11/18/11 | Facile and general synthesis of photoactivatable xanthene dyes.
Wysocki LM, Grimm JB, Tkachuk AN, Brown TA, Betzig E, Lavis LD
Angewandte Chemie. 2011 Nov 18;50:11206-9. doi: 10.1002/anie.201104571

Despite the apparent simplicity of the xanthene fluorophores, the preparation of caged derivatives with free carboxy groups remains a synthetic challenge. A straightforward and flexible strategy for preparing rhodamine and fluorescein derivatives was developed using reduced, “leuco” intermediates.

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11/01/11 | Pupil-segmentation-based adaptive optical microscopy with full-pupil illumination.
Milkie DE, Betzig E, Ji N
Optics Letters. 2011 Nov 1;36(21):4206-8. doi: 10.1364/OL.36.004206

Optical aberrations deteriorate the performance of microscopes. Adaptive optics can be used to improve imaging performance via wavefront shaping. Here, we demonstrate a pupil-segmentation based adaptive optical approach with full-pupil illumination. When implemented in a two-photon fluorescence microscope, it recovers diffraction-limited performance and improves imaging signal and resolution.

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05/01/11 | Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination.
Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, Betzig E
Nature Methods. 2011 May;8(5):417-23. doi: 10.1038/nmeth.1586

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to \~{}0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

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05/01/11 | Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. (With commentary)
Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, Betzig E
Nature Methods. 2011 May;8(5):417-23. doi: 10.1038/nmeth.1586

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to \~{}0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

Commentary: Plane illumination microscopy has proven to be a powerful tool for studying multicellular organisms and their development at single cell resolution. However, the light sheets employed are usually too thick to provide much benefit for imaging organelles within single cultured cells. Here we introduce the use of scanned Bessel beams to create much thinner light sheets better suited to long-term dynamic live cell imaging. Such light sheets not only minimize photobleaching and phototoxicity at the sub-cellular level, but also provide axial resolution enhancement, yielding isotropic three dimensional spatial resolution. Numerous movies are provided to demonstrate the wealth of 4D information (x,y,x,t) that can be obtained from single living cells by the method. Besides providing an attractive alternative to spinning disk, AOD-driven, or line scan confocal microscopes for high speed live cell imaging, the Bessel microscope might serve as a valuable platform for superresolution microscopy (PALM, structured Illumination, or RESOLFT), since confinement of the excitation to the focal plane makes far better use of the limited fluorescence photon budget than does the traditional epi-illumination configuration.

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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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