Joerg Bewersdorf

Yale University, New Haven, Connecticut, United States

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Publications (78)422.99 Total impact

  • 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. · 3.18 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
  • 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
  • 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 · 6.27 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 · 3.73 Impact Factor
  • Biochimica et Biophysica Acta (BBA) - Bioenergetics 07/2014; 1837:e30. DOI:10.1016/j.bbabio.2014.05.305 · 4.83 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: http://dx.doi.org/10.7554/eLife.02758.001
    eLife Sciences 05/2014; 3:e02758. DOI:10.7554/eLife.02758 · 8.52 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 · 20.06 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 · 25.95 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: http://dx.doi.org/10.7554/eLife.01607.001
    eLife Sciences 02/2014; 3:e01607. DOI:10.7554/eLife.01607 · 8.52 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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    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
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    ABSTRACT: A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI: http://dx.doi.org/10.7554/eLife.01296.001
    eLife Sciences 10/2013; 2:e01296. DOI:10.7554/eLife.01296 · 8.52 Impact Factor
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    ABSTRACT: The application of two-photon activation of photoactivatable fluorescent proteins is limited by a lack of information about two-photon activation rates. Here we present rates for the commonly used photoactivatable proteins PAmCherry, PAmKate and PA-GFP at different wavelengths using a novel method that allows us to determine the two-photon activation rates directly, independent of any reference data, with microscopic sample volumes. We show that PAmCherry features the highest rates of the tested proteins at 700 nm activation wavelength followed by PAmKate. Towards longer wavelengths, two-photon activation rates decrease for all three proteins. For PAmCherry, our data contradicts an activation model relying solely on two-photon activation and suggests additional activation pathways requiring at least two absorption steps. Our method is readily expandable to other photoactivatable fluorescent molecules. The presented results allow optimization of experimental conditions in spectroscopic and imaging techniques such as super-resolution fluorescence microscopy.
    Physical Chemistry Chemical Physics 07/2013; 15(36). DOI:10.1039/c3cp51035b · 4.20 Impact Factor
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    ABSTRACT: Stimulated emission depletion (STED) microscopy provides diffraction-unlimited resolution in fluorescence microscopy. Imaging at the nanoscale, however, requires precise alignment of the depletion and excitation laser foci of the STED microscope. We demonstrate here that adaptive optics can be implemented to automatically align STED and confocal images with a precision of 4.3±2.3 nm.
    Optics Letters 06/2013; 38(11):1860-2. DOI:10.1364/OL.38.001860 · 3.18 Impact Factor
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    ABSTRACT: Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.
    Nature Methods 05/2013; 10(7). DOI:10.1038/nmeth.2488 · 25.95 Impact Factor

Publication Stats

2k Citations
422.99 Total Impact Points

Institutions

  • 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
  • 2012
    • International Neuroscience Institute
      Hanover, Lower Saxony, Germany
  • 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