James H. Werner

Los Alamos National Laboratory, Los Alamos, California, United States

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Publications (70)297.12 Total impact

  • [Show abstract] [Hide abstract]
    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). · 10.44 Impact Factor
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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. · 3.39 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. · 3.55 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 04/2014; · 10.44 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.
    02/2014;
  • 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.
    02/2014;
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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. · 2.88 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.
    CLEO: QELS_Fundamental Science; 06/2013
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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; · 5.70 Impact Factor
  • Biophysical Journal 01/2013; 104(2):552-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):545-. · 3.67 Impact Factor
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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. · 13.03 Impact Factor
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    ABSTRACT: We demonstrate following individual fluorescent protein constructs and individual organic dyes as they diffuse in 3-D in solution at rates up to 1 μm(2)/s over distances of several micrometers in X, Y, and Z. Our 3-D tracking method is essentially a stage scanning confocal microscope that uses a unique spatial filter geometry and active feedback 200 times/s to follow fast 3-D motion. Here we detail simulations used to find optimal feedback parameters for following individual fluorescent proteins in 3-D and show that a wide range of parameters are capable of following individual proteins diffusing at 1 μm(2)/s rates. In addition, we experimentally show that through 3-D single-molecule tracking of a protein oligomer series (monomer, dimer, and tetramer) of the fluorescent protein Azami Green one can determine the protein oligomerization state. We also perform time-resolved spectroscopy (photon pair correlation measurements) during the measured 3-D trajectories. The photon pair correlation measurements show clear fluorescence photon antibunching, demonstrating that the trajectories are of single fluorescent molecules. We note that the rates of single-molecule diffusive motion we follow (approximately 1 μm(2)/s) are comparable to or faster than many intracellular transport processes.
    ACS Nano 09/2012; 6(10):8922-32. · 12.03 Impact Factor
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    ABSTRACT: Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individual's genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a different alignment between the Ag nanocluster and the enhancer. This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Samples with single-nucleotide variations can be easily identified by the naked eye under UV excitation, making this method a reliable and low-cost assay with a simple readout format.
    Journal of the American Chemical Society 07/2012; 134(28):11550-8. · 10.68 Impact Factor
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    ABSTRACT: Conjugated systems are frequently studied in their nanoaggregate form to probe the effects of solvent and of film formation on their spectral and dynamical properties. This article focuses on the emission spectra and dynamics of nanoaggregates of alkoxy-substituted PPV oligomers with the goal of interpreting the vibronic emission envelopes observed in these systems (J. Phys. Chem. C2009, 113, 18851-18862). The aggregates are formed by adding a nonsolvent such as methanol (MeOH) or water to a solution of the oligomers in a good solvent such as methyl tetrahydrofuran (MeTHF) or tetrahydrofuran (THF). The emission spectra of aggregates formed using either of these combinations exhibit a vibronic pattern in which the ratio of the intensity of highest-energy band to that of the lower energy peaks depends strongly on the ratio of good to poor solvent. In aggregates formed from MeTHF:MeOH, this was shown to be due to the presence of both aggregate-like and monomer-like emitters forming a "core" and surrounding "shell"-like structure, respectively, within a single aggregate (J. Phys. Chem. C2011, 115, 15607-15616). In support of this model, the monomer-like emission is shown here to be significantly decreased by changing the solvent pair to the more polar THF:water. This suggests that nanoaggregates formed in THF:water contain a much smaller proportion of monomer-like chains than those formed in MeTHF/MeOH, as would be expected from using a more highly polar nonsolvent. Results from bulk steady-state and time-resolved emission measurements as well as fluorescence lifetime imaging microscopy (FLIM) of the aggregates are shown to be consistent with this interpretation.
    The Journal of Physical Chemistry B 06/2012; 116(35):10504-13. · 3.61 Impact Factor
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    ABSTRACT: Metal nanoclusters have interesting steady state fluorescence emission, two-photon excited emission and ultrafast dynamics. A new subclass of fluorescent silver nanoclusters (Ag NCs) are NanoCluster Beacons. NanoCluster Beacons consist of a weakly emissive Ag NC templated on a single stranded DNA ("Ag NC on ssDNA") that becomes highly fluorescent when a DNA enhancer sequence is brought in proximity to the Ag NC by DNA base pairing ("Ag NC on dsDNA"). Steady state fluorescence was observed at 540 nm for both Ag NC on ssDNA and dsDNA; emission at 650 nm is observed for Ag NC on dsDNA. The emission at 550 nm is eight times weaker than that at 650 nm. Fluorescence up-conversion was used to study the dynamics of the emission. Bi-exponential fluorescence decay was recorded at 550 nm with lifetimes of 1 ps and 17 ps. The emission at 650 nm was not observed at the time scale investigated but has been reported to have a lifetime of 3.48 ns. Two-photon excited fluorescence was detected for Ag NC on dsDNA at 630 nm when excited at 800 nm. The two-photon absorption cross-section was calculated to be ∼3000 GM. Femtosecond transient absorption experiments were performed to investigate the excited state dynamics of DNA-Ag NC. An excited state unique to Ag NC on dsDNA was identified at ∼580 nm as an excited state bleach that related directly to the emission at 650 nm based on the excitation spectrum. Based on the optical results, a simple four level system is used to describe the emission mechanism for Ag NC on dsDNA.
    Nanoscale 06/2012; 4(14):4247-54. · 6.73 Impact Factor
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    ABSTRACT: We report the discovery of a DNA sequence that templates a highly stable fluorescent silver nanocluster. In contrast to other DNA templated silver nanoclusters that have a relatively short shelf-life, the fluorescent species templated in this new DNA sequence retains significant fluorescence for at least a year. Moreover, this new silver nanocluster possesses low cellular toxicity and enhanced thermal, oxidative, and chemical stability.
    Nanoscale 05/2012; 4(14):4107-10. · 6.73 Impact Factor
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    ABSTRACT: Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.
    Proc SPIE 02/2012;
  • Biophysical Journal 01/2012; 102(3):722-. · 3.67 Impact Factor
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    ABSTRACT: The electronic properties of single-walled carbon nanotubes can be altered by surface adsorption of electronic impurities or dopants. However, fully understanding the influence of these impurities is difficult because of the inherent complexity of the solution-based colloidal chemistry of nanotubes, and because of a lack of techniques for directly imaging dynamic processes involving these impurities. Here, we show that photoluminescence microscopy can be used to image exciton quenching in semiconducting single-walled carbon nanotubes during the early stages of chemical doping with two different species. The addition of AuCl(3) leads to localized exciton-quenching sites, which are attributed to a mid-gap electronic impurity level, and the adsorbed species are also found sometimes to be mobile on the surface of the nanotubes. The addition of H(2)O(2) leads to delocalized exciton-quenching hole states, which are responsible for long-range photoluminescence blinking, and are also mobile.
    Nature Nanotechnology 01/2012; 7(2):126-32. · 31.17 Impact Factor

Publication Stats

612 Citations
297.12 Total Impact Points

Institutions

  • 2000–2014
    • Los Alamos National Laboratory
      • • Center for Integrated Nanotechnologies
      • • Bioscience Division
      Los Alamos, California, United States
  • 2012
    • University of Texas at Austin
      Austin, Texas, United States