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12 Janelia Publications

Showing 1-10 of 12 results
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    04/26/19 | Dynamic super-resolution structured illumination imaging in the living brain.
    Turcotte R, Liang Y, Tanimoto M, Zhang Q, Li Z, Koyama M, Betzig E, Ji N
    Proceedings of the National Academy of Sciences of the United States of America. 2019 Apr 26;116(19):9586-91. doi: 10.1073/pnas.1819965116

    Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

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    06/10/16 | in vivo brain imaging with adaptive optical microscope.
    Wang K, Sun W, Ji N, Betzig E
    Conference on Lasers and Electro-Optics (CLEO): Applications and Technology. 2016 Jun :AM40.1. doi: 10.1364/CLEO_AT.2016.AM4O.1

    The diffraction limited resolution of two photon and confocal microscope can be recovered using adaptive optics to explore the detailed neuronal network in the brains of zebrafish and mouse in vivo.

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    06/15/15 | Direct wavefront sensing for high-resolution in vivo imaging in scattering tissue.
    Wang K, Sun W, Richie CT, Harvey BK, Betzig E, Ji N
    Nature Communications. 2015-Jun-15;6:7276. doi: 10.1038/ncomms8276

    Adaptive optics by direct imaging of the wavefront distortions of a laser-induced guide star has long been used in astronomy, and more recently in microscopy to compensate for aberrations in transparent specimens. Here we extend this approach to tissues that strongly scatter visible light by exploiting the reduced scattering of near-infrared guide stars. The method enables in vivo two-photon morphological and functional imaging down to 700 μm inside the mouse brain.

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    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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    11/01/11 | 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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    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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    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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    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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    12/01/08 | Advances in the speed and resolution of light microscopy. (With commentary)
    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.

    Commentary: A brief review of recent trends in microscopy. The section “Caveats regarding the application of superresolution microscopy” was written in an effort to inject a dose of reality and caution into the unquestioning enthusiasm in the academic community for all things superresolution, covering the topics of labeling density and specificity, sample preparation artifacts, speed vs. resolution vs. photodamage, and the implications of signal-to-background for Nyquist vs. Rayleigh definitions of resolution.

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