James H. Werner

Los Alamos National Laboratory, Лос-Аламос, California, United States

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Publications (78)357.27 Total impact

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    ABSTRACT: Single particle tracking has provided a wealth of information about biophysical processes such as motor protein transport and diffusion in cell membranes. However, motion out of the plane of the microscope or blinking of the fluorescent probe used as a label generally limits observation times to several seconds. Here, we overcome these limitations by using novel non-blinking quantum dots as probes and employing a custom 3D tracking microscope to actively follow motion in three dimensions (3D) in live cells. Signal-to-noise is improved in the cellular milieu through the use of pulsed excitation and time-gated detection.
    Proc SPIE 03/2015; 9338. DOI:10.1117/12.2082943
  • P Zhang · P M Goodwin · J H Werner ·
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    ABSTRACT: Interferometric detection of the fluorescence emission from a single molecule [interferometric photoactivated localization microscopy (iPALM)] enables a localization accuracy of nanometers in axial localization for 3D superresolution imaging. However, iPALM uses two high-numerical-aperture (NA) objectives in juxtaposition for fluorescence collection (a 4Pi microscope geometry), increasing expense and limiting samples that can be studied. Here, we propose an interferometric single molecule localization microscopy method using a single high-NA objective. The axial position of single molecules can be unambiguously determined from the phase-shifted interference signals with nanometer precision and over a range of 2λ. The use of only one objective simplifies the system configuration and sample mounting. In addition, due to the use of wavefront-splitting interference in our approach, the two parts of the wavefront that eventually merge and interfere with each other travel along nearly equivalent optical paths, which should minimize the effect of drift for long-term 3D superresolution imaging.
    Applied Optics 11/2014; 53(31):7415-7421. DOI:10.1364/AO.53.007415 · 1.78 Impact Factor
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    ABSTRACT: While semiconductor quantum dots (QDs) have been used successfully in numerous single particle tracking (SPT) studies due to their high photoluminescence efficiency, photostability, and broad palette of emission colors, conventional QDs exhibit fluorescence intermittency or ‘blinking,’ which causes ambiguity in particle trajectory analysis and limits tracking duration. Here, non-blinking ‘giant’ quantum dots (gQDs) are exploited to study IgE-FcεRI receptor dynamics in live cells using a confocal-based 3D SPT microscope. There is a 7-fold increase in the probability of observing IgE-FcεRI for longer than 1 min using the gQDs compared to commercially available QDs. A time-gated photon-pair correlation analysis is implemented to verify that selected SPT trajectories are definitively from individual gQDs and not aggregates. The increase in tracking duration for the gQDs allows the observation of multiple changes in diffusion rates of individual IgE-FcεRI receptors occurring on long (>1 min) time scales, which are quantified using a time-dependent diffusion coefficient and hidden Markov modeling. Non-blinking gQDs should become an important tool in future live cell 2D and 3D SPT studies, especially in cases where changes in cellular dynamics are occurring on the time scale of several minutes.
    Advanced Functional Materials 08/2014; 24(30). DOI:10.1002/adfm.201400349 · 11.81 Impact Factor
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    ABSTRACT: Single particle tracking (SPT) of receptors in live cells is typically limited by the intermittency of the fluorescent probe's emission (blinking) or by motion of the receptor out of the imaging plane of the microscope. These limitations are overcome by J. A. Hollingsworth, J. H. Werner, and co‐workers by integrating non‐blinking ‘giant’ quantum dots (gQDs) for confocal‐based 3D live cell SPT of the IgEFcRI allergen receptor. On page 4796, this allows an extended tracking duration compared to conventional core/shell blinking QDs, leading to observations of heterogeneous receptor diffusion occurring over time scales of minutes.
    Advanced Functional Materials 08/2014; 24(30). DOI:10.1002/adfm.201470200 · 11.81 Impact Factor
  • P Zhang · M E Phipps · P M Goodwin · J H Werner ·
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    ABSTRACT: We have developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples with inexpensive visible lasers and a single galvo mirror (GM). The light-sheet is created by raster scanning a Bessel beam with a GM, with this same GM also being used to rescan the fluorescence across a chip of a camera to construct an image in real time. A slit is used to reject out-of-focus fluorescence such that the image formed in real time has minimal contribution from the sidelobes of the Bessel beam. Compared with two-photon Bessel beam excitation or other confocal line-scanning approaches, our method is of lower cost, is simpler, and does not require calibration and synchronization of multiple GMs. We demonstrated the optical sectioning and out-of-focus background rejection capabilities of this microscope by imaging fluorescently labeled actin filaments in fixed 3T3 cells.
    Optics Letters 06/2014; 39(12):3682-3685. DOI:10.1364/OL.39.003682 · 3.29 Impact Factor
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    P Zhang · P M Goodwin · J H Werner ·
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    ABSTRACT: A substantial advantage of stimulated emission depletion (STED) microscopy over other super-resolution methods is that images can be acquired in real-time without any post-processing. However imaging speed and photodamage are two major concerns for STED imaging of whole cells. Here we propose a new microscopy method we have termed Bessel-Beam STED (or BB-STED) that overcomes both of these limitations of conventional STED microscopy. In the proposed method, rather than exciting a single STED spot in the sample, an entire line of the sample is illuminated. This line-scanning technique dramatically increases the speed of STED. In addition, plane-illumination by scanning of the line across the focal plane of a detection objective limits the light to a thin layer of the sample and thus significantly reduces photobleaching and photodamage above and below the focal plane compared to epi-illumination. Using the organic dye Atto647N as an example, we calculated the STED power required to break the diffraction limit. The results presented here will be used to guide future experimental designs.
    Optics Express 05/2014; 22(10):12398-12409. DOI:10.1364/OE.22.012398 · 3.49 Impact Factor
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    ABSTRACT: Here we present an automated microscope capable of 3D multi-color single molecule localization of individual messenger RNA molecules in a wide range of cell types. We have implemented astigmatic imaging with a cylindrical lens to improve z-localization, and a maximum likelihood estimator on a graphics processing unit to improve localization precision and speed. This microscope will aid in gene expression analysis by its capability to perform high throughput imaging of thick cells and tissues while still maintaining sufficient z resolution to resolve single RNA transcripts in three dimensions. Enhanced z-localization allows for resolving membrane localized and co-localized transcripts.
    Proceedings of SPIE - The International Society for Optical Engineering 02/2014; 8950. DOI:10.1117/12.2038197 · 0.20 Impact Factor
  • Pengfei Zhang · Peter M. Goodwin · James H. Werner ·
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    ABSTRACT: We developed a light-sheet illumination microscope that can perform fast 3D imaging of transparent biological samples. The light-sheet is created by raster scanning of a Bessel Beam with one galvo-mirror. Fluorescence excited from the thin layer of the sample is de-scanned by the same galvo-mirror, and then spatially filtered by a slit so that out-of-focus fluorescence generated by the side lobes of the Bessel beam is rejected. The spatially filtered fluorescence is returned by a series of optics and is re-scanned by the same galvo-mirror across the chip of a camera such that the fluorescence image is constructed in real-time. Compared to two-photon Bessel beam excitation or other confocal line scanning approaches, our method is of lower cost, simpler, and doesn't require calibration and synchronization of multiple galvo mirrors. We demonstrated the capability of fast 3D imaging and background rejection capabilities of this microscope with fluorescent beads embedded in PDMS.
    Proceedings of SPIE - The International Society for Optical Engineering 02/2014; 8947. DOI:10.1117/12.2036848 · 0.20 Impact Factor
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    Biophysical Journal 01/2014; 106(2):194a. DOI:10.1016/j.bpj.2013.11.1147 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):374a. DOI:10.1016/j.bpj.2013.11.2118 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):216a. DOI:10.1016/j.bpj.2013.11.1266 · 3.97 Impact Factor
  • Jason J Han · Yuliya A Kunde · Elizabeth Hong-Geller · James H Werner ·
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    ABSTRACT: We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.
    Journal of Biomedical Optics 01/2014; 19(1):16011. DOI:10.1117/1.JBO.19.1.016011 · 2.86 Impact Factor
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    ABSTRACT: We describe the development and characterization of two novel non-blinking "giant" nanocrystal quantum dot (g-NQD) systems and their application to single-particle tracking in live cells real-time and in three dimensions. The two g-NQDs - CdSe/CdS and InP/CdS - are characterized by a thick shell layer (>10 monolayers of CdS), a quasi-type-II or type-II electronic structure, and extremely suppressed non-radiative Auger recombination and photobleaching, in addition to suppressed blinking. We show that these unique traits make this new class of NQD ideal molecular probes for observing spatio-temporal dynamics of cellular processes.
    CLEO: QELS_Fundamental Science; 06/2013
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    ABSTRACT: DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors.
    06/2013; 3(2):185-200. DOI:10.3390/bios3020185

  • 223th ECS Meeting; 05/2013
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    ABSTRACT: Here we present a modification to single-molecule fluorescence in-situ hybridization (smFISH) that enables quantitative detection and analysis of small RNA (sRNA) expressed in bacteria. We show that short (~200 nucleotide) nucleic acid targets can be detected when the background of unbound singly-dye labeled DNA oligomers is reduced through hybridization with a set of complementary DNA oligomers labeled with a fluorescence quencher. By neutralizing the fluorescence from unbound probes, we were able to significantly reduce the number of false positives, allowing for accurate quantification of sRNA levels. Exploiting an automated, mutli-color wide-field microscope and data analysis package, we analyzed the statistics of sRNA expression in thousands of individual bacteria. We found that only a small fraction of either Yersinia pseudotuberculosis or Yersinia pestis bacteria express the small RNAs YSR35 or YSP8, with the copy number typically between 1-10 transcripts. The numbers of these RNA are both increased (by a factor of 2.5x for YSR35 and 3.5x for YSP8) upon a temperature shift from 25°C to 37°C, suggesting they play a role in pathogenesis. The copy number distribution of sRNAs from bacteria-to-bacteria are well-fit with a bursting model of gene transcription. The ability to directly quantify expression levels changes of sRNA in single cells as a function of external stimuli provides key information on the role of sRNA in cellular regulatory networks.
    Analytical Chemistry 04/2013; 85(10). DOI:10.1021/ac303792p · 5.64 Impact Factor
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    Biophysical Journal 01/2013; 104(2):552-. DOI:10.1016/j.bpj.2012.11.3059 · 3.97 Impact Factor
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    Biophysical Journal 01/2013; 104(2):545-. DOI:10.1016/j.bpj.2012.11.3020 · 3.97 Impact Factor
  • Jason J. Han · Andrew P. Shreve · James H. Werner ·
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    ABSTRACT: Far-field super-resolution optical microscopy is a young and rapidly developing technology that enables optical imaging with unprecedented sub-diffraction-limited spatial resolution. These imaging methods have been developed primarily within the context of biological imaging, but show great potential as a new and complementary tool for characterization of nanostructural material features traditionally inaccessible by conventional optical imaging methods. In this article, we introduce the working principles of super resolution optical microscopy, suggest specific materials that should be good candidates for these new techniques, and discuss practical aspects that should be considered for its implementation for materials characterization.Keywords:super-resolution;optical nanoscopy;stimulated emission depletion microscopy;STED;photoactivated localization microscopy;PALM;structured illumination microscopy;SIM
    Characterization of Materials, 10/2012; , ISBN: 0471266965
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    ABSTRACT: We present measurements of S(1) exciton transport in (6,5) carbon nanotubes at room temperature in a colloidal environment. Exciton diffusion lengths associated with end quenching paired with photoluminescence lifetimes provide a direct basis for determining a median diffusion constant of approximately 7.5 cm(2)s(-1). Our experimental results are compared to model diffusion constants calculated using a realistic exciton dispersion accounting for a logarithmic correction due to the exchange self-energy and a nonequilibrium distribution between bright and dark excitons. The intrinsic diffusion constant associated with acoustic phonon scattering is too large to explain the observed diffusion length, and as such, we attribute the observed transport to disorder-limited diffusional transport associated with the dynamics of the colloidal interface. In this model an effective surface potential limits the exciton mean free path to the same size as that of the exciton wave function, defined by the strength of the electron-hole Coulomb interaction.
    Nano Letters 09/2012; 12(10):5091-6. DOI:10.1021/nl301739d · 13.59 Impact Factor

Publication Stats

2k Citations
357.27 Total Impact Points


  • 1999-2015
    • Los Alamos National Laboratory
      • • Center for Integrated Nanotechnologies
      • • Bioscience Division
      • • Life Sciences Division
      Лос-Аламос, California, United States
  • 2011-2012
    • University of Groningen
      Groningen, Groningen, Netherlands
  • 2001
    • University of Washington Seattle
      • Department of Chemistry
      Seattle, Washington, United States