Joerg Bewersdorf

Yale University, New Haven, Connecticut, United States

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Publications (81)456.26 Total impact

  • Caroline Laplante · Fang Huang · Joerg Bewersdorf · Thomas D. Pollard ·

    01/2016: pages 45-57;
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    ABSTRACT: Efficient use of two-photon excitation (TPE) microscopy requires knowledge of the absolute TPE action cross sections (ATACSs) of fluorescent probes. However, these values are not available for recently developed dyes, which exhibit superior properties in many modern microscopy applications. We report ATACSs of five red to far-red organic dyes, ATTO 647N, STAR 635P, silicon rhodamine, ATTO 594, and ATTO 590. The dyes were found to have large ATACSs (>100 GM) at their respective wavelength peaks, thus supporting their use as bright fluorescent markers in TPE microscopy.
    Optics Letters 10/2015; 40(21):4915-4918. DOI:10.1364/OL.40.004915 · 3.29 Impact Factor
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    ABSTRACT: 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 Physics addresses 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. Mark Bates, Christian Eggeling
    Journal of Physics D Applied Physics 10/2015; 48:443001. DOI:10.1088/0022-3727/48/44/443001 · 2.72 Impact Factor
  • Xiang Hao · Edward S Allgeyer · Martin J Booth · Joerg Bewersdorf ·
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    ABSTRACT: IsoSTED nanoscopy, a variant of stimulated emission depletion (STED) microscopy, utilizes two opposing objective lenses and features the highest three-dimensional resolution of STED nanoscopes currently available. However, this technique is limited by axially repetitive side minima in the interference pattern of the depletion point-spread function (PSF), which can lead to ghost images. Here, we describe novel strategies to further improve the performance of isoSTED nanoscopy by reshaping the PSF. In particular, we propose employing moderate defocus on the depletion beam to reduce the side minima. Furthermore, we demonstrate a simplified alternative based on objective misalignment and quantitatively compare the expected performance between the two approaches.
    Optics Letters 08/2015; 40(15):3627-3630. DOI:10.1364/OL.40.003627 · 3.29 Impact Factor
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    ABSTRACT: Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.
    PLoS ONE 05/2015; 10(5):e0128135. DOI:10.1371/journal.pone.0128135 · 3.23 Impact Factor
  • D. Burke · B. Patton · F. Huang · J. Bewersdorf · M.J. Booth ·
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    ABSTRACT: Specimen-induced aberrations frequently affect image quality in high-resolution microscopes. Aberration effects can be even more problematic in super-resolution methods. We show adaptive aberration correction in STORM microscopy of deep cell and tissue specimens.
  • Daniel Burke · Brian Patton · Fang Huang · Joerg Bewersdorf · Martin J. Booth ·
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    ABSTRACT: Single-molecule switching (SMS) microscopy is a super-resolution method capable of producing images with resolutions far exceeding that of the classical diffraction limit. However, like all optical microscopes, SMS microscopes are sensitive to, and often limited by, specimen-induced aberrations. Adaptive optics (AO) has proven beneficial in a range of microscopes to overcome the limitations caused by aberrations. We report here on new AO methods for SMS microscopy that enable the feedback correction of specimen-induced aberrations. The benefits are demonstrated through two-dimensional and three-dimensional STORM imaging. We expect that this advance will broaden the scope of SMS microscopy by enabling deep-cell and tissue-level imaging.
    Optica 02/2015; 2(2):177. DOI:10.1364/OPTICA.2.000177
  • Fang Huang · Caroline E. Laplante · Yu Lin · Thomas D. Pollard · Joerg Bewersdorf ·

    Biophysical Journal 01/2015; 108(2):475a. DOI:10.1016/j.bpj.2014.11.2599 · 3.97 Impact Factor
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    ABSTRACT: Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.
    PLoS ONE 12/2014; 9(12):e114043. DOI:10.1371/journal.pone.0114043 · 3.23 Impact Factor
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    ABSTRACT: Wir berichten über eine Lipid-basierte Strategie zur Visualisierung von Struktur und Dynamik des Golgi-Apparats in lebenden Zellen mithilfe hochauflösender Mikroskopie. Die Methode basiert auf zwei neuen Reagentien: einem trans-Cycloocten enthaltenden Ceramid-Lipid (Cer-TCO) und einem hoch reaktiven, Tetrazin-markierten Nah-IR-Farbstoff (SiR-Tz). Diese beiden Komponenten reagieren in einer extrem schnellen Tetrazin-Klick-Reaktion zu Cer-SiR, einer sehr photostabilen Verbindung, welche die Visualisierung des Golgi-Apparats sowohl mit 3D-Konfokalmikroskopie als auch mit hochauflösender Mikroskopie über eine längere Zeitspanne ermöglicht. Cer-SiR ist nicht toxisch bis zu einer Konzentration von 2 μM und stört weder die Mobilität von Enzymen innerhalb des Golgi-Apparats noch den Transport von Fracht vom Endoplasmatischen Retikulum durch den Golgi-Apparat zur Plasmamembran.
    Angewandte Chemie 09/2014; 126(38). DOI:10.1002/ange.201403349
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    ABSTRACT: We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid “tetrazine-click” reaction into Cer-SiR, a highly photostable “vital dye” that enables prolonged live-cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is nontoxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.
    Angewandte Chemie International Edition 09/2014; 53(38). DOI:10.1002/anie.201403349 · 11.26 Impact Factor
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    ABSTRACT: Focal adhesions (FAs) are macromolecular complexes that connect the actin cytoskeleton to the extracellular matrix. Dynamic turnover of FAs is critical for cell migration. Paxillin is a multi-adaptor protein that plays an important role in regulating FA dynamics. Here, we identify TRIM15, a member of the TRIpartite Motif protein family, as a paxillin-interacting factor and a component of FAs. TRIM15 localizes to focal contacts in a myosin II-independent manner by an interaction between its coiled coil domain and the LD2 motif of paxillin. Unlike other FA proteins, TRIM15 is a stable FA component with restricted mobility due to its ability to form oligomers. TRIM15-depleted cells display impaired cell migration and FA disassembly rates in addition to enlarged FAs. Thus, our studies demonstrate a cellular function for TRIM15 as a regulatory component of FA turnover and cell migration.
    Development 07/2014; 127(18). DOI:10.1242/jcs.143537 · 6.46 Impact Factor
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    ABSTRACT: Fronto-temporal lobar degeneration with TDP-43 (FTLD-TDP) is a fatal neurodegeneration. TMEM106B variants are linked to FTLD-TDP risk, and TMEM106B is lysosomal. Here, we focus on neuronal TMEM106B, and demonstrate co-localization and traffic with lysosomal LAMP-1. pH-sensitive reporters demonstrate that the TMEM106B C-terminus is lumenal. The TMEM106B N-terminus interacts with endosomal adaptors and other TMEM106 proteins. TMEM106B knockdown reduces neuronal lysosomal number and diameter by STED microscopy, and overexpression enlarges LAMP-positive structures. Reduction of TMEM106B increases axonally transported lysosomes, while TMEM106B elevation inhibits transport and yields large lysosomes in the soma. TMEM106B overexpression alters lysosomal stress signaling, causing a translocation of the mTOR-sensitive transcription factor, TFEB, to neuronal nuclei. TMEM106B loss-of-function delays TFEB translocation after Torin-1-induced stress. Enlarged TMEM106B-overexpressing lysosomes maintain organelle integrity longer after lysosomal photodamage than do control lysosomes, while small TMEM106B-knockdown lysosomes are more sensitive to illumination. Thus, neuronal TMEM106B plays a central role in regulating lysosomal size, motility and responsiveness to stress, highlighting the possible role of lysosomal biology in FTLD-TDP.
    Molecular and Cellular Neuroscience 07/2014; 61. DOI:10.1016/j.mcn.2014.07.006 · 3.84 Impact Factor

  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e30. DOI:10.1016/j.bbabio.2014.05.305 · 5.35 Impact Factor
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    ABSTRACT: The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria. DOI:
    eLife Sciences 05/2014; 3(3):e02758. DOI:10.7554/eLife.02758 · 9.32 Impact Factor
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    ABSTRACT: The components supporting autophagosome growth on the cup-like isolation membrane are likely to be different from those found on closed and maturing autophagosomes. The highly curved rim of the cup may serve as a functionally required surface for transiently associated components of the early acting autophagic machinery. Here we demonstrate that the E2-like enzyme, Atg3, facilitates LC3/GABARAP lipidation only on membranes exhibiting local lipid-packing defects. This activity requires an amino-terminal amphipathic helix similar to motifs found on proteins targeting highly curved intracellular membranes. By tuning the hydrophobicity of this motif, we can promote or inhibit lipidation in vitro and in rescue experiments in Atg3-knockout cells, implying a physiologic role for this stress detection. The need for extensive lipid-packing defects suggests that Atg3 is designed to work at highly curved membranes, perhaps including the limiting edge of the growing phagophore.
    Nature Cell Biology 04/2014; 16(8). DOI:10.1038/ncb2940 · 19.68 Impact Factor
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    ABSTRACT: Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized. We present a lucid synthesis of the developments on this localization precision and accuracy and their practical implications in order to guide the increasing number of researchers using single-particle tracking and super-resolution localization microscopy.
    Nature Methods 02/2014; 11(3):253-66. DOI:10.1038/nmeth.2843 · 32.07 Impact Factor
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    ABSTRACT: Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids, such as triacylglycerol (TG), as reservoirs of metabolic energy and membrane precursors. The Arf1/COPI protein machinery, known for its role in vesicle trafficking, regulates LD morphology, targeting of specific proteins to LDs and lipolysis through unclear mechanisms. Recent evidence shows that Arf1/COPI can bud nano-LDs (∼60 nm diameter) from phospholipid-covered oil/water interfaces in vitro. We show that Arf1/COPI proteins localize to cellular LDs, are sufficient to bud nano-LDs from cellular LDs, and are required for targeting specific TG-synthesis enzymes to LD surfaces. Cells lacking Arf1/COPI function have increased amounts of phospholipids on LDs, resulting in decreased LD surface tension and impairment to form bridges to the ER. Our findings uncover a function for Arf1/COPI proteins at LDs and suggest a model in which Arf1/COPI machinery acts to control ER-LD connections for localization of key enzymes of TG storage and catabolism. DOI:
    eLife Sciences 02/2014; 3(3):e01607. DOI:10.7554/eLife.01607 · 9.32 Impact Factor
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    Joerg Bewersdorf ·

    Biophysical Journal 01/2014; 106(2):6a. DOI:10.1016/j.bpj.2013.11.069 · 3.97 Impact Factor
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    Sheng Liu · Emil B Kromann · Wesley D Krueger · Joerg Bewersdorf · Keith A Lidke ·
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    ABSTRACT: Localization-based superresolution imaging is dependent on finding the positions of individual fluorophores in a sample by fitting the observed single-molecule intensity pattern to the microscope point spread function (PSF). For three-dimensional imaging, system-specific aberrations of the optical system can lead to inaccurate localizations when the PSF model does not account for these aberrations. Here we describe the use of phase-retrieved pupil functions to generate a more accurate PSF and therefore more accurate 3D localizations. The complex-valued pupil function contains information about the system-specific aberrations and can thus be used to generate the PSF for arbitrary defocus. Further, it can be modified to include depth dependent aberrations. We describe the phase retrieval process, the method for including depth dependent aberrations, and a fast fitting algorithm using graphics processing units. The superior localization accuracy of the pupil function generated PSF is demonstrated with dual focal plane 3D superresolution imaging of biological structures.
    Optics Express 12/2013; 21(24):29462-87. DOI:10.1364/OE.21.029462 · 3.49 Impact Factor

Publication Stats

2k Citations
456.26 Total Impact Points


  • 2010-2015
    • Yale University
      • • Department of Cell Biology
      • • Department of Biomedical Engineering
      New Haven, Connecticut, United States
  • 2010-2014
    • Yale-New Haven Hospital
      New Haven, Connecticut, United States
  • 1998-2010
    • Max Planck Institute for Biophysical Chemistry
      • Department of NanoBiophotonics
      Göttingen, Lower Saxony, Germany
  • 2007-2009
    • University of Maine
      • • Department of Physics and Astronomy
      • • Department Mathematics & Statistics
      Orono, Minnesota, United States
    • The Jackson Laboratory
      Bar Harbor, Maine, United States