Bio

Bio


W. E. (William Esco) Moerner, the Harry S. Mosher Professor of Chemistry and Professor, by courtesy, of Applied Physics at Stanford University, has conducted research in the areas of physical chemistry and biophysics of single molecules and is actively involved in the development of 2D and 3D super-resolution imaging for cell biology. Imaging studies include protein superstructures in bacteria, structure of huntingtin protein aggregates, centriole proteins, and ion channel distributions. Using a powerful microscope optimized for tracking of single objects in cells, the motions of DNA and RNA are being measured in three dimensions in real time to understand processing and binding interactions. A related research area concerns precise analysis of photodynamics of single trapped biomolecules in solution, with applications to photosynthesis, electron transport catalysis, and diffusion/mobility transport measurements.

Academic Appointments


Administrative Appointments


  • Chairman, Department of Chemistry, Stanford University (2011 - 2014)
  • Member, Board of Scientific Counselors, NIBIB (2010 - 2014)
  • Member, Advisory Board, Center for Biological Imaging at Stanford (2010 - Present)
  • Chair, University Health and Safety Committee (2008 - 2010)

Honors & Awards


  • John Gamble Kirkwood Medal, New Haven Section, American Chemical Society (2013)
  • Engineering Alumni Achievement Award, Washington University (2013)
  • Peter Debye Award in Physical Chemistry, American Chemical Society (2013)
  • Pittsburgh Award in Spectroscopy, PittCon (2012)
  • Irving Langmuir Prize in Chemical Physics, American Physical Society (2009)
  • Wolf Prize in Chemistry, Wolf Foundation of Israel (2008)
  • Member, National Academy of Sciences (2007)
  • Earle K. Plyler Prize in Molecular Spectroscopy, American Physical Society (2001)

Boards, Advisory Committees, Professional Organizations


  • Member, Board of Scientific Counselors, NIBIB (2010 - Present)
  • Member, Advisory Board, Institute of Atomic and Molecular Sciences, Academica Sinica, Taiwan (2003 - Present)

Professional Education


  • Ph.D., Cornell University, Physics (1982)
  • M.S., Cornell University, Physics (1978)
  • B. S., Washington University, Physics (1975)
  • B. S., Washington University, Electrical Engineering (1975)
  • A. B., Washington University, Mathematics (1975)

Research & Scholarship

Current Research and Scholarly Interests


Most biophysical or chemical experiments in condensed matter measure the average behavior of a huge number, N, of molecules, where N may range from millions to billions to Avogadro's Number. At the same time, most theoretical models are intended to describe the behavior of a single molecule interacting with its surroundings, and averaging over the number of molecules N is normally required to compute an observable. Using precision laser spectroscopic techniques, we have been detecting and probing the detailed properties of individual impurity molecules hidden deep inside a cell, in a protein, or even in a liquid, i.e., the ultimate limit of N=1. This was first done in the Moerner Lab in 1989, and has since expanded dramatically to include many groups around the world. A key reason for doing this is to explore heterogeneity that is normally obscured by ensemble averaging.

Studying one individual molecule in a solid means we are working with an extremely small number of moles of material. You might be aware that the international standards organization, IUPAC, has defined several new prefixes: zepto- for 1E-21, and yocto- for 1E-24. Thus 1 molecule is equivalent to 1.66 yoctomoles. But we think this is unwieldy. Thus we define a new prefix guaca- so that (with apologies to Prof. Avogadro)

1 guacamole = 1 / ( Avocado's Number) of moles.

More seriously, it is worth recalling that each molecule we are probing is only 1 or 2 nanometers in size. This means that when we use a laser to select one probe molecule, we can sense details of the immediate local environment of a truly nanoscopic probe.

To achieve this extreme reduction of the concentration and reach the single-molecule level, we use either (a) extremely low concentrations and diffraction-limited confocal, TIRF, or far-field microscopy, or (b) near field optical excitation to pump sample volumes much smaller than the diffraction limit, or (c) superresolution imaging by single-molecule active control. By studying a large number of individual molecules one at a time, we are able not only to observe how the usual ensemble average behavior is formed, but also to see unexpected, surprising behavior normally hidden by the usual ensemble averaging.

The phenomena under study include protein localization patternd in bacteria, chaperonin proteins, and new fluorophores for active-control superresolution imaging. By dispersing the emitted light, even the vibrational mode spectrum of a single molecule may be measured! By measuring correlations in the emitted photon stream, fast dynamics including environmental fluctuations, or the purely quantum-mechanical behavior termed photon antibunching may be probed. In biomolecules, we observe fascinating differences in behavior due to conformational states, local environments, or enzymatic cycle, all of which are obscured in large N experiments.

Importantly, a single molecule can be viewed as a probe of its immediate local nanoenvironment on the scale on the order of the molecular size (~1 nm). Because single molecules are nanoscale emitters, when active control is used to turn molecules on and off, it is possible to build up a super-resolution image of the sample, far beyond the optical diffraction limit, typically on the 40 nm scale. Several advanced optical techniques for obtaining thee-dimensional information from single-molecule photoswitching are underdevelopment, and we apply these methods to imaging a variety of cellular structures in bacteria and in mammalian cells and to tracking of RNA in living yeast.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nature reviews. Microbiology Gahlmann, A., Moerner, W. E. 2013; 12 (1): 9-22

    Abstract

    The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

    View details for DOI 10.1038/nrmicro3154

    View details for PubMedID 24336182

  • Quantifying Transient 3D Dynamical Phenomena of Single mRNA Particles in Live Yeast Cell Measurements JOURNAL OF PHYSICAL CHEMISTRY B Calderon, C. P., Thompson, M. A., Casolari, J. M., Paffenroth, R. C., Moerner, W. E. 2013; 117 (49): 15701-15713

    Abstract

    Single-particle tracking (SPT) has been extensively used to obtain information about diffusion and directed motion in a wide range of biological applications. Recently, new methods have appeared for obtaining precise (10s of nm) spatial information in three dimensions (3D) with high temporal resolution (measurements obtained every 4 ms), which promise to more accurately sense the true dynamical behavior in the natural 3D cellular environment. Despite the quantitative 3D tracking information, the range of mathematical methods for extracting information about the underlying system has been limited mostly to mean-squared displacement analysis and other techniques not accounting for complex 3D kinetic interactions. There is a great need for new analysis tools aiming to more fully extract the biological information content from in vivo SPT measurements. High-resolution SPT experimental data has enormous potential to objectively scrutinize various proposed mechanistic schemes arising from theoretical biophysics and cell biology. At the same time, methods for rigorously checking the statistical consistency of both model assumptions and estimated parameters against observed experimental data (i.e., goodness-of-fit tests) have not received great attention. We demonstrate methods enabling (1) estimation of the parameters of 3D stochastic differential equation (SDE) models of the underlying dynamics given only one trajectory; and (2) construction of hypothesis tests checking the consistency of the fitted model with the observed trajectory so that extracted parameters are not overinterpreted (the tools are applicable to linear or nonlinear SDEs calibrated from nonstationary time series data). The approach is demonstrated on high-resolution 3D trajectories of single ARG3 mRNA particles in yeast cells in order to show the power of the methods in detecting signatures of transient directed transport. The methods presented are generally relevant to a wide variety of 2D and 3D SPT tracking applications.

    View details for DOI 10.1021/jp4064214

    View details for Web of Science ID 000328529000047

    View details for PubMedID 24015725

  • Super-resolution fluorescence imaging with single molecules. Current opinion in structural biology Sahl, S. J., Moerner, W. 2013; 23 (5): 778-787

    Abstract

    The ability to detect, image and localize single molecules optically with high spatial precision by their fluorescence enables an emergent class of super-resolution microscopy methods which have overcome the longstanding diffraction barrier for far-field light-focusing optics. Achieving spatial resolutions of 20-40nm or better in both fixed and living cells, these methods are currently being established as powerful tools for minimally-invasive spatiotemporal analysis of structural details in cellular processes which benefit from enhanced resolution. Briefly covering the basic principles, this short review then summarizes key recent developments and application examples of two-dimensional and three-dimensional (3D) multi-color techniques and faster time-lapse schemes. The prospects for quantitative imaging - in terms of improved ability to correct for dipole-emission-induced systematic localization errors and to provide accurate counts of molecular copy numbers within nanoscale cellular domains - are discussed.

    View details for DOI 10.1016/j.sbi.2013.07.010

    View details for PubMedID 23932284

  • Rotational mobility of single molecules affects localization accuracy in super-resolution fluorescence microscopy. Nano letters Lew, M. D., Backlund, M. P., Moerner, W. E. 2013; 13 (9): 3967-3972

    Abstract

    The asymmetric nature of single-molecule (SM) dipole emission patterns limits the accuracy of position determination in localization-based super-resolution fluorescence microscopy. The degree of mislocalization depends highly on the rotational mobility of SMs; only for SMs rotating within a cone half angle ? > 60° can mislocalization errors be bounded to ?10 nm. Simulations demonstrate how low or high rotational mobility can cause resolution degradation or distortion in super-resolution reconstructions.

    View details for DOI 10.1021/nl304359p

    View details for PubMedID 23360306

  • Photo-induced conformational flexibility in single solution-phase peridinin-chlorophyll-proteins. journal of physical chemistry. A Bockenhauer, S. D., Moerner, W. E. 2013; 117 (35): 8399-8406

    Abstract

    The peridinin-chlorophyll-protein (PCP) is an accessory light-harvesting complex found in red-tide dinoflagellates. PCP absorbs photons primarily in the blue-green spectral region via peridinin (Per) carotenoid pigments which then transfer excitations to chlorophyll (Chl) and ultimately downstream to photosystem II (PSII). Whereas the ultrafast dynamics of PCP are well-studied, much less is known about slower protein dynamics on time scales of milliseconds and seconds. Previous single-molecule studies of spectral emission and intensity have attached PCP to surfaces, but the native environment of PCP is in the lumen, meaning that a surface-attached environment could perturb its native conformations. To address this concern, we use the anti-Brownian electrokinetic (ABEL) trap to study single PCP monomers in solution for several seconds each. We measure, for the first time, simultaneous single-molecule intensity, lifetime, and spectral emission shifts for each trapped PCP monomer. The rate of reversible spectral redshifts depends linearly on irradiance over a factor of 30, indicating a light-induced mechanism which we attribute to a protein conformational change. Independent of these spectral shifts, our measurements of intensity and lifetime show reversible Chl quenching. In contrast to previous work, we show that this quenching cannot result from isolated photobleaching of Chl. These independent mechanisms arise from distinct conformational changes which maintain relatively stable fluorescence emission.

    View details for DOI 10.1021/jp405790a

    View details for PubMedID 23919352

  • Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schlau-Cohen, G. S., Wang, Q., Southall, J., Cogdell, R. J., Moerner, W. E. 2013; 110 (27): 10899-10903

    Abstract

    Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.

    View details for DOI 10.1073/pnas.1310222110

    View details for Web of Science ID 000321978000022

    View details for PubMedID 23776245

  • Single-molecule orientation measurements with a quadrated pupil OPTICS LETTERS Backer, A. S., Backlund, M. P., Lew, M. D., Moerner, W. E. 2013; 38 (9): 1521-1523

    Abstract

    This Letter presents a means of measuring the dipole orientation of a fluorescent, orientationally fixed single molecule, which uses a specially designed phase mask, termed a "quadrated pupil," conjugate to the back focal plane of a conventional wide-field microscope. The method leverages the spatial anisotropy of the far-field emission pattern of a dipole emitter and makes this anisotropy amenable to quantitative analysis at the image plane. In comparison to older image-fitting techniques that infer orientation by matching simulations to defocused or excessively magnified images, the quadrated pupil approach is more robust to minor modeling discrepancies and optical aberrations. Precision of 1°-5° is achieved in proof-of-concept experiments for both azimuthal (?) and polar (?) angles without defocusing. Since the phase mask is implemented on a liquid-crystal spatial light modulator that may be deactivated without any mechanical perturbation of the sample or imaging system, the technique may be readily integrated into clear aperture imaging studies.

    View details for DOI 10.1364/OL.38.001521

    View details for Web of Science ID 000318425600054

    View details for PubMedID 23632538

  • Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions NANO LETTERS Gahlmann, A., Ptacin, J. L., Grover, G., Quirin, S., von Diezmann, A. R., Lee, M. K., Backlund, M. P., Shapiro, L., Piestun, R., Moerner, W. E. 2013; 13 (3): 987-993

    Abstract

    We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

    View details for DOI 10.1021/nl304071h

    View details for Web of Science ID 000316243800020

    View details for PubMedID 23414562

  • The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation SINGLE MOLECULE SPECTROSCOPY AND SUPERRESOLUTION IMAGING VI Backlund, M. P., Lew, M. D., Backer, A. S., Sahl, S. J., Grover, G., Agrawal, A., Piestun, R., Moerner, W. E. 2013; 8590

    View details for DOI 10.1117/12.2001671

    View details for Web of Science ID 000321741600013

  • Enzymatic activation of nitro-aryl fluorogens in live bacterial cells for enzymatic turnover-activated localization microscopy CHEMICAL SCIENCE Lee, M. K., Williams, J., Twieg, R. J., Rao, J., Moerner, W. E. 2013; 4 (1): 220-225

    View details for DOI 10.1039/c2sc21074f

    View details for Web of Science ID 000311971500023

  • Probing Single Biomolecules in Solution Using the Anti-Brownian Electrokinetic (ABEL) Trap ACCOUNTS OF CHEMICAL RESEARCH Wang, Q., Goldsmith, R. H., Jiang, Y., Bockenhauer, S. D., Moerner, W. E. 2012; 45 (11): 1955-1964

    Abstract

    Single-molecule fluorescence measurements allow researchers to study asynchronous dynamics and expose molecule-to-molecule structural and behavioral diversity, which contributes to the understanding of biological macromolecules. To provide measurements that are most consistent with the native environment of biomolecules, researchers would like to conduct these measurements in the solution phase if possible. However, diffusion typically limits the observation time to approximately 1 ms in many solution-phase single-molecule assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the observed heterogeneity. Combining the technical capabilities of high-sensitivity single-molecule fluorescence microscopy, real-time feedback control and electrokinetic flow in a microfluidic chamber, we have developed a device called the anti-Brownian electrokinetic (ABEL) trap to significantly prolong the observation time of single biomolecules in solution. We have applied the ABEL trap method to explore the photodynamics and enzymatic properties of a variety of biomolecules in aqueous solution and present four examples: the photosynthetic antenna allophycocyanin, the chaperonin enzyme TRiC, a G protein-coupled receptor protein, and the blue nitrite reductase redox enzyme. These examples illustrate the breadth and depth of information which we can extract in studies of single biomolecules with the ABEL trap. When confined in the ABEL trap, the photosynthetic antenna protein allophycocyanin exhibits rich dynamics both in its emission brightness and its excited state lifetime. As each molecule discontinuously converts from one emission/lifetime level to another in a primarily correlated way, it undergoes a series of state changes. We studied the ATP binding stoichiometry of the multi-subunit chaperonin enzyme TRiC in the ABEL trap by counting the number of hydrolyzed Cy3-ATP using stepwise photobleaching. Unlike ensemble measurements, the observed ATP number distributions depart from the standard cooperativity models. Single copies of detergent-stabilized G protein-coupled receptor proteins labeled with a reporter fluorophore also show discontinuous changes in emission brightness and lifetime, but the various states visited by the single molecules are broadly distributed. As an agonist binds, the distributions shift slightly toward a more rigid conformation of the protein. By recording the emission of a reporter fluorophore which is quenched by reduction of a nearby type I Cu center, we probed the enzymatic cycle of the redox enzyme nitrate reductase. We determined the rate constants of a model of the underlying kinetics through an analysis of the dwell times of the high/low intensity levels of the fluorophore versus nitrite concentration.

    View details for DOI 10.1021/ar2003041

    View details for Web of Science ID 000311858300013

    View details for PubMedID 22616716

  • Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species SCIENTIFIC REPORTS Sahl, S. J., Weiss, L. E., Duim, W. C., Frydman, J., Moerner, W. E. 2012; 2

    Abstract

    The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates -100?nm in diameter and up to -1-2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.

    View details for DOI 10.1038/srep00895

    View details for Web of Science ID 000311891000001

    View details for PubMedID 23193437

  • Simultaneous, accurate measurement of the 3D position and orientation of single molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Backlund, M. P., Lew, M. D., Backer, A. S., Sahl, S. J., Grover, G., Agrawal, A., Piestun, R., Moerner, W. E. 2012; 109 (47): 19087-19092

    Abstract

    Recently, single molecule-based superresolution fluorescence microscopy has surpassed the diffraction limit to improve resolution to the order of 20 nm or better. These methods typically use image fitting that assumes an isotropic emission pattern from the single emitters as well as control of the emitter concentration. However, anisotropic single-molecule emission patterns arise from the transition dipole when it is rotationally immobile, depending highly on the molecule's 3D orientation and z position. Failure to account for this fact can lead to significant lateral (x, y) mislocalizations (up to ?50-200 nm). This systematic error can cause distortions in the reconstructed images, which can translate into degraded resolution. Using parameters uniquely inherent in the double-lobed nature of the Double-Helix Point Spread Function, we account for such mislocalizations and simultaneously measure 3D molecular orientation and 3D position. Mislocalizations during an axial scan of a single molecule manifest themselves as an apparent lateral shift in its position, which causes the standard deviation (SD) of its lateral position to appear larger than the SD expected from photon shot noise. By correcting each localization based on an estimated orientation, we are able to improve SDs in lateral localization from ?2× worse than photon-limited precision (48 vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation over different depths, we are able to improve from a lateral SD of 116 (?4× worse than the photon-limited precision; 28 nm) to 34 nm (within 6 nm of the photon limit).

    View details for DOI 10.1073/pnas.1216687109

    View details for Web of Science ID 000311997200022

    View details for PubMedID 23129640

  • Fluorescence correlation spectroscopy at high concentrations using gold bowtie nanoantennas CHEMICAL PHYSICS Kinkhabwala, A. A., Yu, Z., Fan, S., Moerner, W. E. 2012; 406: 3-8
  • Fluorescent Saxitoxins for Live Cell Imaging of Single Voltage-Gated Sodium Ion Channels beyond the Optical Diffraction Limit CHEMISTRY & BIOLOGY Ondrus, A. E., Lee, H. D., Iwanaga, S., Parsons, W. H., Andresen, B. M., Moerner, W. E., Du Bois, J. 2012; 19 (7): 902-912

    Abstract

    A desire to better understand the role of voltage-gated sodium channels (Na(V)s) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select Na(V) isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to Na(V)s in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective Na(V) labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal Na(V) distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.

    View details for DOI 10.1016/j.chembiol.2012.05.021

    View details for Web of Science ID 000307261100016

    View details for PubMedID 22840778

  • STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein BIOPHYSICAL JOURNAL Lau, L., Lee, Y. L., Sahl, S. J., Stearns, T., Moerner, W. E. 2012; 102 (12): 2926-2935

    Abstract

    Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ?300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.

    View details for DOI 10.1016/j.bpj.2012.05.015

    View details for Web of Science ID 000305546500027

    View details for PubMedID 22735543

  • Analytical Tools To Distinguish the Effects of Localization Error, Confinement, and Medium Elasticity on the Velocity Autocorrelation Function BIOPHYSICAL JOURNAL Weber, S. C., Thompson, M. A., Moerner, W. E., Spakowitz, A. J., Theriot, J. A. 2012; 102 (11): 2443-2450

    Abstract

    Single particle tracking is a powerful technique for investigating the dynamic behavior of biological molecules. However, many of the analytical tools are prone to generate results that can lead to mistaken interpretations of the underlying transport process. Here, we explore the effects of localization error and confinement on the velocity autocorrelation function, C?. We show that calculation of C? across a range of discretizations can distinguish the effects of localization error, confinement, and medium elasticity. Thus, under certain regimes, C? can be used as a diagnostic tool to identify the underlying mechanism of anomalous diffusion. Finally, we apply our analysis to experimental data sets of chromosomal loci and RNA-protein particles in Escherichia coli.

    View details for DOI 10.1016/j.bpj.2012.03.062

    View details for Web of Science ID 000305003100006

    View details for PubMedID 22713559

  • Microscopy beyond the diffraction limit using actively controlled single molecules JOURNAL OF MICROSCOPY Moerner, W. E. 2012; 246 (3): 213-220

    Abstract

    In this short review, the general principles are described for obtaining microscopic images with resolution beyond the optical diffraction limit with single molecules. Although it has been known for several decades that single-molecule emitters can blink or turn on and off, in recent work the addition of on/off control of molecular emission to maintain concentrations at very low levels in each imaging frame combined with sequential imaging of sparse subsets has enabled the reconstruction of images with resolution far below the optical diffraction limit. Single-molecule active control microscopy provides a powerful window into information about nanoscale structures that was previously unavailable.

    View details for DOI 10.1111/j.1365-2818.2012.03600.x

    View details for Web of Science ID 000303993700001

    View details for PubMedID 22582796

  • The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision APPLIED PHYSICS LETTERS Lee, H. D., Sahl, S. J., Lew, M. D., Moerner, W. E. 2012; 100 (15)

    View details for DOI 10.1063/1.3700446

    View details for Web of Science ID 000303128000085

  • Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism CHEMPHYSCHEM Biteen, J. S., Goley, E. D., Shapiro, L., Moerner, W. E. 2012; 13 (4): 1007-1012

    Abstract

    Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. We quantify these dynamics and determine the FtsZ depolymerization time to be <100 ms. We image the Z-ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre-divisional stage.

    View details for DOI 10.1002/cphc.201100686

    View details for Web of Science ID 000301537300020

    View details for PubMedID 22262316

  • Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae PLOS ONE Casolari, J. M., Thompson, M. A., Salzman, J., Champion, L. M., Moerner, W. E., Brown, P. O. 2012; 7 (2)

    Abstract

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe here a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.

    View details for DOI 10.1371/journal.pone.0031912

    View details for Web of Science ID 000302796200110

    View details for PubMedID 22359641

  • A Selenium Analogue of Firefly D-Luciferin with Red-Shifted Bioluminescence Emission ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Conley, N. R., Dragulescu-Andrasi, A., Rao, J., Moerner, W. E. 2012; 51 (14): 3350-3353

    Abstract

    A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.

    View details for DOI 10.1002/anie.201105653

    View details for Web of Science ID 000302059400009

    View details for PubMedID 22344705

  • Spectrally Resolved Anti-Brownian ELectrokinetic (ABEL) Trapping of Single Peridinin-Chlorophyll-Proteins in Solution BIOPHOTONICS: PHOTONIC SOLUTIONS FOR BETTER HEALTH CARE III Bockenhauer, S. D., Wang, Q., Moerner, W. E. 2012; 8427

    View details for DOI 10.1117/12.970546

    View details for Web of Science ID 000305701200091

  • Anti-Brownian ELectrokinetic (ABEL) Trapping of Single beta(2)-Adrenergic Receptors in the Absence and Presence of Agonist SINGLE MOLECULE SPECTROSCOPY AND SUPERRESOLUTION IMAGING V Bockenhauer, S., Fuerstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. 2012; 8228

    View details for DOI 10.1117/12.910018

    View details for Web of Science ID 000305624900003

  • Extending Microscopic Resolution with Single-Molecule Imaging and Active Control ANNUAL REVIEW OF BIOPHYSICS, VOL 41 Thompson, M. A., Lew, M. D., Moerner, W. E. 2012; 41: 321-342

    Abstract

    Superresolution imaging of biological structures provides information beyond the optical diffraction limit. One class of superresolution techniques uses the power of single fluorescent molecules as nanoscale emitters of light combined with emission control, variously described by the acronyms PALM/FPALM/STORM and many others. Even though the acronyms differ and refer mainly to different active-control mechanisms, the underlying fundamental principles behind these "pointillist" superresolution imaging techniques are the same. Circumventing the diffraction limit requires two key steps. The first step (superlocalization) is the detection and localization of spatially separated single molecules. The second step actively controls the emitting molecules to ensure a very low concentration of single emitters such that they do not overlap in any one imaging frame. The final image is reconstructed from time-sequential imaging and superlocalization of the single emitting labels decorating the structure of interest. The statistical, imaging, and active-control strategies for achieving superresolution imaging with single molecules are reviewed.

    View details for DOI 10.1146/annurev-biophys-050511-102250

    View details for Web of Science ID 000307955100016

    View details for PubMedID 22577822

  • Conformational dynamics of single G protein-coupled receptors in solution. journal of physical chemistry. B Bockenhauer, S., Fürstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. 2011; 115 (45): 13328-13338

    Abstract

    G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single ?(2)-adrenergic receptors (?(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled ?(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single ?(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent ? ~ 0.5, suggesting protein dynamics over a range of time scales.

    View details for DOI 10.1021/jp204843r

    View details for PubMedID 21928818

  • Conformational Dynamics of Single G Protein-Coupled Receptors in Solution JOURNAL OF PHYSICAL CHEMISTRY B Bockenhauer, S., Fuerstenberg, A., Yao, X. J., Kobilka, B. K., Moerner, W. E. 2011; 115 (45): 13328-13338

    View details for DOI 10.1021/jp204843r

    View details for Web of Science ID 000296686000025

  • Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lew, M. D., Lee, S. F., Ptacin, J. L., Lee, M. K., Twieg, R. J., Shapiro, L., Moerner, W. E. 2011; 108 (46): E1102-E1110

    Abstract

    Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 12 ?m).

    View details for DOI 10.1073/pnas.1114444108

    View details for Web of Science ID 000297008900003

    View details for PubMedID 22031697

  • Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goldsmith, R. H., Tabares, L. C., Kostrz, D., Dennison, C., Aartsma, T. J., Canters, G. W., Moerner, W. E. 2011; 108 (42): 17269-17274

    Abstract

    Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.

    View details for DOI 10.1073/pnas.1113572108

    View details for Web of Science ID 000295975300016

    View details for PubMedID 21969548

  • Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jiang, Y., Douglas, N. R., Conley, N. R., Miller, E. J., Frydman, J., Moerner, W. E. 2011; 108 (41): 16962-16967

    Abstract

    In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

    View details for DOI 10.1073/pnas.1112244108

    View details for Web of Science ID 000295973800024

    View details for PubMedID 21896715

  • Sub-Diffraction Imaging of Huntingtin Protein Aggregates by Fluorescence Blink-Microscopy and Atomic Force Microscopy CHEMPHYSCHEM Duim, W. C., Chen, B., Frydman, J., Moerner, W. E. 2011; 12 (13): 2386-2389
  • Sub-diffraction imaging of huntingtin protein aggregates by fluorescence blink-microscopy and atomic force microscopy. Chemphyschem Duim, W. C., Chen, B., Frydman, J., Moerner, W. E. 2011; 12 (13): 2387-2390

    View details for DOI 10.1002/cphc.201100392

    View details for PubMedID 21735512

  • An Adaptive Anti-Brownian Electrokinetic Trap with Real-Time Information on Single-Molecule Diffusivity and Mobility ACS NANO Wang, Q., Moerner, W. E. 2011; 5 (7): 5792-5799

    Abstract

    We present the design and implementation of an adaptive Anti-Brownian ELectrokinetic (ABEL) trap capable of extracting estimates of the diffusion coefficient and mobility of single trapped fluorescent nanoscale objects such as biomolecules in solution. The system features rapid acousto-optic scanning of a confocal excitation spot on a 2D square lattice to encode position information on the arrival time of each detected photon, and Kalman filter-based signal processing unit for refined position estimation. We demonstrate stable trapping of multisubunit proteins (D ? 22 ?m(2)/s) with a count rate of 6 kHz for as long as 15 s and small single-stranded DNA molecules (D ? 118 ?m(2)/s) at a 15 kHz count rate for seconds. Moreover, we demonstrate real-time measurement of diffusion coefficient and electrokinetic mobility of trapped objects, using adaptive tuning of the Kalman filter parameters.

    View details for DOI 10.1021/nn2014968

    View details for Web of Science ID 000293035200057

    View details for PubMedID 21612271

  • Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus BIOPHYSICAL JOURNAL Lee, S. F., Thompson, M. A., Schwartz, M. A., Shapiro, L., Moerner, W. E. 2011; 100 (7): L31-L33

    Abstract

    Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.

    View details for DOI 10.1016/j.bpj.2011.02.022

    View details for Web of Science ID 000289494200001

    View details for PubMedID 21463569

  • Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects OPTICS LETTERS Lew, M. D., Lee, S. F., Badieirostami, M., Moerner, W. E. 2011; 36 (2): 202-204

    Abstract

    We describe the corkscrew point spread function (PSF), which can localize objects in three dimensions throughout a 3.2 ?m depth of field with nanometer precision. The corkscrew PSF rotates as a function of the axial (z) position of an emitter. Fisher information calculations show that the corkscrew PSF can achieve nanometer localization precision with limited numbers of photons. We demonstrate three-dimensional super-resolution microscopy with the corkscrew PSF by imaging beads on the surface of a triangular polydimethylsiloxane (PDMS) grating. With 99,000 photons detected, the corkscrew PSF achieves a localization precision of 2.7 nm in x, 2.1 nm in y, and 5.7 nm in z.

    View details for Web of Science ID 000286188100036

    View details for PubMedID 21263500

  • Exploring protein superstructures and dynamics in live bacterial cells using single-molecule and superresolution imaging. Methods in molecular biology (Clifton, N.J.) Biteen, J. S., Shapiro, L., Moerner, W. E. 2011; 783: 139-158

    Abstract

    Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the optical diffraction limit, and enables superresolution reconstruction of structures beyond the diffraction limit. This work summarizes how single-molecule and superresolution imaging can be applied to the study of protein dynamics and superstructures in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.

    View details for DOI 10.1007/978-1-61779-282-3_8

    View details for PubMedID 21909887

  • Live-cell single-molecule and superresolution imaging of proteins in bacteria SINGLE MOLECULE SPECTROSCOPY AND IMAGING IV Biteen, J. S., Moerner, W. E. 2011; 7905

    View details for DOI 10.1117/12.873809

    View details for Web of Science ID 000297674400014

  • Azido Push-Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels JOURNAL OF PHYSICAL CHEMISTRY B Lord, S. J., Lee, H. D., Samuel, R., Weber, R., Liu, N., Conley, N. R., Thompson, M. A., Twieg, R. J., Moerner, W. E. 2010; 114 (45): 14157-14167

    Abstract

    Dark azido push-pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push-pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push-pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push-pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling).

    View details for DOI 10.1021/jp907080r

    View details for Web of Science ID 000284018000006

    View details for PubMedID 19860443

  • Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H. D., Lord, S. J., Iwanaga, S., Zhan, K., Xie, H., Williams, J. C., Wang, H., Bowman, G. R., Goley, E. D., Shapiro, L., Twieg, R. J., Rao, J., Moerner, W. E. 2010; 132 (43): 15099-15101

    Abstract

    Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

    View details for DOI 10.1021/ja1044192

    View details for Web of Science ID 000283621700003

    View details for PubMedID 20936809

  • Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thompson, M. A., Casolari, J. M., Badieirostami, M., Brown, P. O., Moerner, W. E. 2010; 107 (42): 17864-17871

    Abstract

    Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25-nm precision in the x and y dimensions and 50-nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types.

    View details for DOI 10.1073/pnas.1012868107

    View details for Web of Science ID 000283184800008

    View details for PubMedID 20921361

  • Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane APPLIED PHYSICS LETTERS Badieirostami, M., Lew, M. D., Thompson, M. A., Moerner, W. E. 2010; 97 (16)

    View details for DOI 10.1063/1.3499652

    View details for Web of Science ID 000283502100003

  • Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET JOURNAL OF MOLECULAR BIOLOGY Kim, S. Y., Miller, E. J., Frydman, J., Moerner, W. E. 2010; 401 (4): 553-563

    Abstract

    The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

    View details for DOI 10.1016/j.jmb.2010.06.050

    View details for Web of Science ID 000281262400001

    View details for PubMedID 20600107

  • A spindle-like apparatus guides bacterial chromosome segregation NATURE CELL BIOLOGY Ptacin, J. L., Lee, S. F., Garner, E. C., Toro, E., Eckart, M., Comolli, L. R., Moerner, W., Shapiro, L. 2010; 12 (8): 791-U46

    Abstract

    Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

    View details for DOI 10.1038/ncb2083

    View details for Web of Science ID 000280561600011

    View details for PubMedID 20657594

  • Single-Molecule Spectroscopy and Imaging of Biomolecules in Living Cells ANALYTICAL CHEMISTRY Lord, S. J., Lee, H. D., Moerner, W. E. 2010; 82 (6): 2192-2203

    Abstract

    The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. This Perspective summarizes the field of live-cell imaging of single biomolecules.

    View details for DOI 10.1021/ac9024889

    View details for Web of Science ID 000275379300007

    View details for PubMedID 20163145

  • Single-Molecule and Superresolution Imaging in Live Bacteria Cells COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Biteen, J. S., Moerner, W. E. 2010; 2 (3)

    Abstract

    Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.

    View details for DOI 10.1101/cshperspect.a000448

    View details for Web of Science ID 000279881700002

    View details for PubMedID 20300204

  • Watching conformational- and photodynamics of single fluorescent proteins in solution NATURE CHEMISTRY Goldsmith, R. H., Moerner, W. E. 2010; 2 (3): 179-186

    Abstract

    Observing the dynamics of single biomolecules over prolonged time periods is difficult to achieve without significantly altering the molecule through immobilization. It can, however, be accomplished using the Anti-Brownian ELectrokinetic (ABEL) Trap, which allows extended investigation of solution-phase biomolecules - without immobilization -through real-time electrokinetic feedback. Here we apply the ABEL trap to study an important photosynthetic antenna protein, Allophycocyanin (APC). The technique allows the observation of single molecules of solution-phase APC for more than one second. We observe a complex relationship between fluorescence intensity and lifetime that cannot be explained by simple static kinetic models. Light-induced conformational changes are shown to occur and evidence is obtained for fluctuations in the spontaneous emission lifetime, which is typically assumed to be constant. Our methods provide a new window into the dynamics of fluorescent proteins and the observations are relevant for the interpretation of in vivo single-molecule imaging experiments, bacterial photosynthetic regulation, and biomaterials for solar energy harvesting.

    View details for DOI 10.1038/NCHEM.545

    View details for Web of Science ID 000274648800012

    View details for PubMedID 20625479

  • Localizing and Tracking Single Emitters in Three Dimensions Using a Double-Helix Point Spread Function 2010 CONFERENCE ON LASERS AND ELECTRO-OPTICS (CLEO) AND QUANTUM ELECTRONICS AND LASER SCIENCE CONFERENCE (QELS) Thompson, M. A., Lew, M. D., Badieirostami, M., Moerner, W. E. 2010
  • In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function. Proceedings - Society of Photo-Optical Instrumentation Engineers Lew, M. D., Thompson, M. A., Badieirostami, M., Moerner, W. E. 2010; 7571: 75710Z

    Abstract

    The point spread function (PSF) of a widefield fluorescence microscope is not suitable for three-dimensional super-resolution imaging. We characterize the localization precision of a unique method for 3D superresolution imaging featuring a double-helix point spread function (DH-PSF). The DH-PSF is designed to have two lobes that rotate about their midpoint in any transverse plane as a function of the axial position of the emitter. In effect, the PSF appears as a double helix in three dimensions. By comparing the Cramer-Rao bound of the DH-PSF with the standard PSF as a function of the axial position, we show that the DH-PSF has a higher and more uniform localization precision than the standard PSF throughout a 2 ?m depth of field. Comparisons between the DH-PSF and other methods for 3D super-resolution are briefly discussed. We also illustrate the applicability of the DH-PSF for imaging weak emitters in biological systems by tracking the movement of quantum dots in glycerol and in live cells.

    View details for PubMedID 20563317

  • Localizing and Tracking Single Nanoscale Emitters in Three Dimensions with High Spatiotemporal Resolution Using a Double-Helix Point Spread Function NANO LETTERS Thompson, M. A., Lew, M. D., Badieirostami, M., Moerner, W. E. 2010; 10 (1): 211-218

    Abstract

    Three-dimensional nanoscale localization and tracking of dim single emitters can be obtained with a widefield fluorescence microscope exhibiting a double-helix point spread function (DH-PSF). We describe in detail how the localization precision quantitatively depends upon the number of photons detected and the z position of the nanoscale emitter, thereby showing a approximately 10 nm localization capability along x, y, and z in the limit of weak emitters. Experimental measurements are compared to Fisher information calculations of the ultimate localization precision inherent in the DH-PSF. The DH-PSF, for the first time, is used to track single quantum dots in aqueous solution and a quantum dot-labeled structure inside a living cell in three dimensions.

    View details for DOI 10.1021/nl903295p

    View details for Web of Science ID 000273428700036

    View details for PubMedID 20000821

  • Optimal strategy for trapping single fluorescent molecules in solution using the ABEL trap. Applied physics. B, Lasers and optics Wang, Q., Moerner, W. E. 2010; 99 (1-2): 23-30

    Abstract

    Trapping of 10-nm-sized single fluorescent bio-molecules in solution has been achieved using high-speed position sensing and electrokinetic feedback forces in the Anti-Brownian ELectrokinetic (ABEL) trap. The high diffusion coefficient of small objects in solution requires very fast, real-time sensing of position, and this has been previously achieved using a simple rotating beam, but improved strategies are needed for the smallest objects, such as single nanometer-sized fluorescent molecules. At the same time, single molecules are limited in photon emission rate and total number of photons, so each emitted photon must be used as efficiently as possible. We describe a new controller design for the ABEL trap which features fast, knight's tour scanning of an excitation beam on a 2D square lattice and a Kalman filter-based estimator for optimal position sensing. This strategy leads directly to a maximum-likelihood-based method to extract the diffusion coefficient of the object held in the trap. The effectiveness of the algorithms are demonstrated and compared to the simple rotating beam design through Monte Carlo simulations. Our new approach yields tighter trapping and a much improved ability to extract diffusion coefficients.

    View details for PubMedID 20383275

  • MOLECULES AND METHODS FOR SUPER-RESOLUTION IMAGING METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Thompson, M. A., Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E. 2010; 475: 27-59

    Abstract

    By looking at a fluorescently labeled structure one molecule at a time, it is possible to side-step the optical diffraction limit and obtain "super-resolution" images of small nanostructures. In the Moerner Lab, we seek to develop both molecules and methods to extend super-resolution fluorescence imaging. Methodologies and protocols for designing and characterizing fluorophores with switchable fluorescence required for super-resolution imaging are reported. These fluorophores include azido-DCDHF molecules, covalently linked Cy3-Cy5 dimers, and also the first example of a photoswitchable fluorescent protein, enhanced yellow fluorescent protein (EYFP). The imaging of protein superstructures in living Caulobacter crescentus bacteria is used as an example of the power of super-resolution imaging by single-molecule photoswitching to extract information beyond the diffraction limit. Finally, a new method is described for obtaining three-dimensional super-resolution information using a double-helix point-spread function.

    View details for DOI 10.1016/S0076-6879(10)75002-3

    View details for Web of Science ID 000280733800002

    View details for PubMedID 20627152

  • In vivo Three-Dimensional Superresolution Fluorescence Tracking using a Double-Helix Point Spread Function SINGLE MOLECULE SPECTROSCOPY AND IMAGING III Lew, M. D., Thompson, M. A., Badieirostami, M., Moerner, W. E. 2010; 7571

    View details for DOI 10.1117/12.842608

    View details for Web of Science ID 000284353600015

  • Micrometer-Sized DNA-Single-Fluorophore-DNA Supramolecule: Synthesis and Single-Molecule Characterization SMALL Lee, J. K., Jaeckel, F., Moerner, W. E., Bao, Z. 2009; 5 (21): 2418-2423

    Abstract

    The synthesis of single-fluorophore-bis(micrometer-sized DNA) triblock supramolecules and the optical and structural characterization of the construct at the single-molecule level is reported. A fluorophore-bis(oligodeoxynucleotide) triblock is synthesized via the amide-coupling reaction. Subsequent protocols of DNA hybridization/ligation are developed to form the supramolecular triblock structure with lambda-DNA fragments on the micrometer length scale. The successful synthesis of the micrometer-sized DNA-single-fluorophore-DNA supramolecule is confirmed by agarose gel electrophoresis with fluorescence imaging under UV excitation. Single triblock structures are directly imaged by combined scanning force microscopy and single-molecule fluorescence microscopy, and provide unambiguous confirmation of the existence of the single fluorophore inserted in the middle of the long DNA. This type of triblock structure is a step closer to providing a scaffold for single-molecule electronic devices after metallization of the DNAs.

    View details for DOI 10.1002/smll.200900494

    View details for Web of Science ID 000271791100011

    View details for PubMedID 19517486

  • Large single-molecule fluorescence enhancements produced by a bowtie nanoantenna NATURE PHOTONICS Kinkhabwala, A., Yu, Z., Fan, S., Avlasevich, Y., Muellen, K., Moerner, W. E. 2009; 3 (11): 654-657
  • Lithographic positioning of fluorescent molecules on high-Q photonic crystal cavities APPLIED PHYSICS LETTERS Rivoire, K., Kinkhabwala, A., Hatami, F., Masselink, W. T., Avlasevich, Y., Muellen, K., Moerner, W. E., Vuckovic, J. 2009; 95 (12)

    View details for DOI 10.1063/1.3232233

    View details for Web of Science ID 000270243800065

  • Bright, Red Single-Molecule Emitters: Synthesis and Properties of Environmentally Sensitive Dicyanomethylenedihydrofuran (DCDHF) Fluorophores with Bisaromatic Conjugation CHEMISTRY OF MATERIALS Lu, Z., Liu, N., Lord, S. J., Bunge, S. D., Moerner, W. E., Twieg, R. J. 2009; 21 (5): 797-810

    View details for DOI 10.1021/cm801783f

    View details for Web of Science ID 000263891700007

  • Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pavani, S. R., Thompson, M. A., Biteen, J. S., Lord, S. J., Liu, N., Twieg, R. J., Piestun, R., Moerner, W. E. 2009; 106 (9): 2995-2999

    Abstract

    We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10- to 20-nm precision over a large depth of field (2 microm) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.

    View details for DOI 10.1073/pnas.0900245106

    View details for Web of Science ID 000263844100007

    View details for PubMedID 19211795

  • DCDHF Fluorophores for Single-Molecule Imaging in Cells CHEMPHYSCHEM Lord, S. J., Conley, N. R., Lee, H. D., Nishimura, S. Y., Pomerantz, A. K., Willets, K. A., Lu, Z., Wang, H., Liu, N., Samuel, R., Weber, R., Semyonov, A., He, M., Twieg, R. J., Moerner, W. E. 2009; 10 (1): 55-65

    Abstract

    There is a persistent need for small-molecule fluorescent labels optimized for single-molecule imaging in the cellular environment. Application of these labels comes with a set of strict requirements: strong absorption, efficient and stable emission, water solubility and membrane permeability, low background emission, and red-shifted absorption to avoid cell autofluorescence. We have designed and characterized several fluorophores, termed "DCDHF" fluorophores, for use in live-cell imaging based on the push-pull design: an amine donor group and a 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) acceptor group, separated by a pi-rich conjugated network. In general, the DCDHF fluorophores are comparatively photostable, sensitive to local environment, and their chemistries and photophysics are tunable to optimize absorption wavelength, membrane affinity, and solubility. Especially valuable are fluorophores with sophisticated photophysics for applications requiring additional facets of control, such as photoactivation. For example, we have reengineered a red-emitting DCDHF fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. This molecule and its relatives provide a new class of bright photoactivatable small-molecule fluorophores, which are needed for super-resolution imaging schemes that require active control (here turning-on) of single-molecule emission.

    View details for DOI 10.1002/cphc.200800581

    View details for Web of Science ID 000262870500010

    View details for PubMedID 19025732

  • Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable Enhanced Yellow Fluorescent Protein SINGLE MOLECULE SPECTROSCOPY AND IMAGING II Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L., Moerner, W. E. 2009; 7185

    View details for DOI 10.1117/12.809080

    View details for Web of Science ID 000285712600011

  • Photoactivatable DCDHF fluorophores for single-molecule imaging REPORTERS, MARKERS, DYES, NANOPARTICLES, AND MOLECULAR PROBES FOR BIOMEDICAL APPLICATIONS Lord, S. J., Conley, N. R., Lee, H. D., Liu, N., Samuel, R., Twieg, R. J., Moerner, W. E. 2009; 7190

    View details for DOI 10.1117/12.809257

    View details for Web of Science ID 000285710200021

  • Probing High-Q Photonic Crystal Resonances With Fluorescent Molecules 2009 CONFERENCE ON LASERS AND ELECTRO-OPTICS AND QUANTUM ELECTRONICS AND LASER SCIENCE CONFERENCE (CLEO/QELS 2009), VOLS 1-5 Rivoire, K., Kinkhabwala, A., Moerner, W. E., Vuckovic, J., Hatami, F., Masselink, W. T., Avlasevich, Y., Muellen, K. 2009: 2353-2354
  • Three-Dimensional Super-resolution Single-Molecule Fluorescence Imaging Using a Double-Helix Point Spread Function 2009 CONFERENCE ON LASERS AND ELECTRO-OPTICS AND QUANTUM ELECTRONICS AND LASER SCIENCE CONFERENCE (CLEO/QELS 2009), VOLS 1-5 Thompson, M. A., Biteen, J. S., Moerner, W. E., Pavani, S. R., Piestun, R. 2009: 1921-1922
  • Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP NATURE METHODS Biteen, J. S., Thompson, M. A., Tselentis, N. K., Bowman, G. R., Shapiro, L., Moerner, W. E. 2008; 5 (11): 947-949

    Abstract

    The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.

    View details for DOI 10.1038/nmeth.1258

    View details for Web of Science ID 000260532500013

    View details for PubMedID 18794860

  • Visualization of Long Human Telomere Mimics by Single-Molecule Fluorescence Imaging JOURNAL OF PHYSICAL CHEMISTRY B Pomerantz, A. K., Moerner, W. E., Kool, E. T. 2008; 112 (42): 13184-13187

    Abstract

    Study of long single-stranded telomeric DNA is important for a variety of basic science and biotechnological applications, yet few methods exist for synthesis and visualization of single copies of this DNA in solution at biologically relevant length scales necessary for assessment of heterogeneity in its structure and behavior. We have synthesized kilobase-long single-stranded human telomere mimics in situ by rolling circle replication (RCR) on a microscope coverslip surface and visualized individual strands by staining with SYBR Gold. Under buffer flow, differential extensibility and varying morphology of these long telomere-mimicking DNA sequences were observed at the single-molecule level in real time. Using this procedure, we detected striking differences in the extensibility of individual RCR products based on the human G-rich telomeric sequence in the presence and absence of short, complementary single-stranded oligonucleotides. We also apply this new mode of single-stranded DNA characterization to probe the interaction of kilobase-length telomere mimics with the small-molecule G-quadruplex-binding agent TMPyP4.

    View details for DOI 10.1021/jp806696u

    View details for Web of Science ID 000260100900005

    View details for PubMedID 18817431

  • Cy3-Cy5 covalent heterodimers for single-molecule photoswitching JOURNAL OF PHYSICAL CHEMISTRY B Conley, N. R., Biteen, J. S., Moerner, W. E. 2008; 112 (38): 11878-11880

    Abstract

    Covalent heterodimers of the Cy3 and Cy5 fluorophores have been prepared from commercially available starting materials and characterized at the single-molecule level. This system behaves as a discrete molecular photoswitch, in which photoexcitation of the Cy5 results in fluorescence emission or, with a much lower probability, causes the Cy5 to enter into a long-lived, but metastable, dark state. Photoinduced recovery of the emissive Cy5 is achieved by very low intensity excitation (5 W cm(-2)) of the Cy3 fluorophore at a shorter wavelength. A similar system consisting of proximal, but not covalently linked, Cy3 and Cy5 has found application in stochastic optical reconstruction microscopy (STORM), a single-molecule localization-based technique for super-resolution imaging that requires photoswitching. The covalent Cy3-Cy5 heterodimers described herein eliminate the need for probabilistic methods of situating the Cy3 and Cy5 in close proximity to enable photoswitching. As proof of principle, these heterodimers have been applied to super-resolution imaging of the tubular stalk structures of live Caulobacter crescentus bacterial cells.

    View details for DOI 10.1021/jp806698p

    View details for Web of Science ID 000259342000004

    View details for PubMedID 18754575

  • A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole CELL Bowman, G. R., Comolli, L. R., Zhu, J., Eckart, M., Koenig, M., Downing, K. H., Moerner, W. E., Earnest, T., Shapiro, L. 2008; 134 (6): 945-955

    Abstract

    Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.

    View details for DOI 10.1016/j.cell.2008.07.015

    View details for Web of Science ID 000259318100015

    View details for PubMedID 18805088

  • Single-molecule motions of oligoarginine transporter conjugates on the plasma membrane of Chinese hamster ovary cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H., Dubikovskaya, E. A., Hwang, H., Semyonov, A. N., Wang, H., Jones, L. R., TWIEG, R. J., Moerner, W. E., Wender, P. A. 2008; 130 (29): 9364-9370

    Abstract

    To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K(+)] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.

    View details for DOI 10.1021/ja710798b

    View details for Web of Science ID 000257796500048

    View details for PubMedID 18578528

  • A photoactivatable push-pull fluorophore for single-molecule imaging in live cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lord, S. J., Conley, N. R., Lee, H. D., Samuel, R., Liu, N., Twieg, R. J., Moerner, W. E. 2008; 130 (29): 9204-?

    Abstract

    We have reengineered a red-emitting dicyanomethylenedihydrofuran push-pull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.

    View details for DOI 10.1021/ja802883k

    View details for Web of Science ID 000257796500013

    View details for PubMedID 18572940

  • Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer OPTICS EXPRESS Cohen, A. E., Moerner, W. E. 2008; 16 (10): 6941-6956

    Abstract

    We present an Anti-Brownian Electrokinetic trap (ABEL trap) capable of trapping individual fluorescently labeled protein molecules in aqueous buffer. The ABEL trap operates by tracking the Brownian motion of a single fluorescent particle in solution, and applying a time-dependent electric field designed to induce an electrokinetic drift that cancels the Brownian motion. The trapping strength of the ABEL trap is limited by the latency of the feedback loop. In previous versions of the trap, this latency was set by the finite frame rate of the camera used for video-tracking. In the present system, the motion of the particle is tracked entirely in hardware (without a camera or image-processing software) using a rapidly rotating laser focus and lock-in detection. The feedback latency is set by the finite rate of arrival of photons. We demonstrate trapping of individual molecules of the protein GroEL in buffer, and we show confinement of single fluorophores of the dye Cy3 in water.

    View details for Web of Science ID 000256469800020

    View details for PubMedID 18545398

  • Superresolution Imaging in Live Bacterial Cells by Single-Molecule Active-Control Microscopy 2008 CONFERENCE ON LASERS AND ELECTRO-OPTICS & QUANTUM ELECTRONICS AND LASER SCIENCE CONFERENCE, VOLS 1-9 Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L., Moerner, W. E. 2008: 246-247
  • Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: Control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes OPTICAL TRAPPING AND OPTICAL MICROMANIPULATION V Jiang, Y., Wang, Q., Cohen, A. E., Douglas, N., Frydman, J., Moerner, W. E. 2008; 7038

    View details for DOI 10.1117/12.798093

    View details for Web of Science ID 000262711000004

  • Gold bowtie nanoantennas for surface-enhanced Raman scattering under controlled electrochemical potential CHEMICAL PHYSICS LETTERS Jackel, F., Kinkhabwala, A. A., Moerner, W. E. 2007; 446 (4-6): 339-343
  • Photophysical properties of acene DCDHF fluorophores: Long-wavelength single-molecule emitters designed for cellular Imaging JOURNAL OF PHYSICAL CHEMISTRY A Lord, S. J., Lu, Z., Wang, H., Willetst, K. A., Schuck, P. J., Lee, H. D., Nishimura, S. Y., Twieg, R. J., Moerner, W. E. 2007; 111 (37): 8934-8941

    Abstract

    We report the solvatochromic, viscosity-sensitive, and single-molecule photophysics of the fluorophores DCDHF-N-6 and DCDHF-A-6. These molecules are members of the dicyanomethylenedihydrofuran (DCDHF) class of single-molecule emitters that contain an amine electron donor and a DCDHF acceptor linked by a conjugated unit; DCDHF-N-6 and DCDHF-A-6 have naphthalene- and anthracene-conjugated linkers, respectively. These molecules maintain the beneficial photophysics of the phenylene-linked DCDHF (i.e., photostability, emission wavelength dependence on solvent polarity, and quantum yield sensitivity to solvent viscosity), yet offer absorption and emission at longer wavelengths that are more appropriate for cellular imaging. We demonstrate that these new fluorophores are less photolabile in an aqueous environment than several other commonly used dyes (rhodamine 6G, Texas Red, and fluorescein). Finally, we image single copies of the acene DCDHFs diffusing in the plasma membrane of living cells.

    View details for DOI 10.1021/jp0712598

    View details for Web of Science ID 000249501600005

    View details for PubMedID 17718454

  • New directions in single-molecule imaging and analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Moerner, W. E. 2007; 104 (31): 12596-12602

    Abstract

    Optical imaging and analysis of single molecules continue to unfold as powerful ways to study the individual behavior of biological systems, unobscured by ensemble averaging. Current expansion of interest in this field is great, as evidenced by new meetings, journal special issues, and the large number of new investigators. Selected recent advances in biomolecular analysis are described, and two new research directions are summarized: superresolution imaging using single-molecule fluorescence and trapping of single molecules in solution by direct suppression of Brownian motion.

    View details for DOI 10.1073/pnas.0610081104

    View details for Web of Science ID 000248603900004

    View details for PubMedID 17664434

  • Principal-components analysis of shape fluctuations of single DNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Moerner, W. E. 2007; 104 (31): 12622-12627

    Abstract

    Thermal fluctuations agitate molecules in solution over a broad range of times and distances. By passively watching the shape fluctuations of a thermally driven biomolecule, one can infer properties of the underlying interactions that determine the motion. We applied this concept to single molecules of fluorescently labeled lambda-DNA, a key model system for polymer physics. In contrast to most other single-molecule DNA experiments, we examined the unstretched, equilibrium state of DNA by using an anti-Brownian electrokinetic trap to confine the center of mass of the DNA without perturbing its internal dynamics. We analyze the long-wavelength conformational normal modes, calculate their spring constants, and measure linear and nonlinear couplings between modes. The modes show strong signs of nonlinear hydrodynamics, a feature of the underlying equations of polymer dynamics that has not previously been reported and is neglected in the widely used Rouse and Zimm approximations.

    View details for DOI 10.1073/pnas.0610396104

    View details for Web of Science ID 000248603900008

    View details for PubMedID 17496147

  • Bulk and single-molecule characterization of an improved molecular beacon utilizing H-dimer excitonic behavior JOURNAL OF PHYSICAL CHEMISTRY B Conley, N. R., Pomerantz, A. K., Wang, H., Twieg, R. J., Moerner, W. E. 2007; 111 (28): 7929-7931

    Abstract

    Pairs of fluorophores in close proximity often show self-quenching of fluorescence by the well-known H-dimer mechanism. We use a pair of fluorophores in the new dicyanomethylenedihydrofuran (DCDHF) dye family in the design and characterization of a new fluorescent probe for nucleic acid detection, which we refer to as a self-quenched intramolecular dimer (SQuID) molecular beacon (MB). We obtain a quenching efficiency of 97.2%, higher than the only other reported value for a MB employing fluorophore self-quenching by H-dimer formation. Furthermore, the excellent single-molecule (SM) emitter characteristics of the DCDHF dyes allow observation of individual SQuID MB-target complexes immobilized on a surface, where the doubled SM emission intensity of our target-bound beacon ensures a higher signal-to-background ratio than conventional fluorophore-quencher MBs. Additional advantages of the SQuID MB are single-pot labeling, visible colorimetric detection of the target, and intrinsic single-molecule two-step photobleaching behavior, which offers a specific means of discriminating between functional MBs and spurious fluorescence.

    View details for DOI 10.1021/jp073310d

    View details for Web of Science ID 000247966300001

    View details for PubMedID 17583944

  • Modifications of DCDHF single molecule fluorophores to impart water solubility TETRAHEDRON LETTERS Wang, H., Lu, Z., Lord, S. J., Moerner, W. E., Twieg, R. J. 2007; 48 (19): 3471-3474
  • Internal mechanical response of a polymer in solution PHYSICAL REVIEW LETTERS Cohen, A. E., Moerner, W. E. 2007; 98 (11)

    Abstract

    We observed single molecules of fluorescently labeled double-stranded (ds) lambda DNA held in an anti-Brownian electrokinetic trap. From the measured density fluctuations we extract the density-density response function of the molecule over times >4.5 ms and distances >250 nm, i.e., how a perturbation in density in one part of the molecule propagates through the rest of the molecule. We find a nonmonotonic radial dependence of the relaxation time. In contrast with earlier measurements on freely diffusing dsDNA, we observe clear signs of internal hydrodynamic interactions.

    View details for DOI 10.1103/PhysRevLett.98.116001

    View details for Web of Science ID 000244959300041

    View details for PubMedID 17501066

  • The influence of tetrahydroquinoline rings in dicyanomethylenedihydrofuran (DCDHF) single-molecule fluorophores TETRAHEDRON Wang, H., Lu, Z., Lord, S. J., Willets, K. A., Bertke, J. A., Bunge, S. D., Moerner, W. E., Twieg, R. J. 2007; 63 (1): 103-114
  • Single-molecule tracking. Methods in molecular biology (Clifton, N.J.) Vrljic, M., Nishimura, S. Y., Moerner, W. E. 2007; 398: 193-219

    Abstract

    The current models of eukaryotic plasma membrane organization separate the plasma membrane nto different environments created by lipids and interactions between membrane proteins and the cytoskeleton, but characterization of their physical properties, such as their sizes, lifetimes, and the partitioning of membrane components into each environment, has not been accomplished. Single-moleule (fluorophore) tracking (SMT) experiments are well suited to the noninvasive study of membrane properties. In SMT experiments, the position of a single fluorescently labeled protein or lipid probe is followed optically as it moves within the membrane. If the motion of the probe is unhindered, then the atial trajectory of the molecule will follow two-dimensional Brownian motion. If the probe encounters a structure that in some way inhibits its movement, then the probe's trajectory will deviate from Brownian motion. It is likely that even if a certain type of lipid or protein partitions strongly into one nvironment, each individual lipid or protein will spend some fraction of its lifetime in the less favorable environment. Because SMT follows the motion of an individual probe over a large area (approximately 10 x 10 microm2), transitions between environments can be observed directly by monitoring the path of each protein or lipid. Additionally, heterogeneity owing to multiple populations of molecules permanently residng in different states may be distinguished from a single population of molecules transitioning between different states. By judicious choice of label, such that the motion of the labeled protein or lipid is unafected by the label itself, and through the use of probes with different affinities for each membrane environment, SMT measurements in principle can reveal the structure of the plasma membrane.

    View details for DOI 10.1007/978-1-59745-513-8_14

    View details for PubMedID 18214382

  • Single-molecule electron spin resonance APPLIED MAGNETIC RESONANCE von Borczyskowski, C., Koehler, J., Moerner, W. E., Orrit, M., Wrachtrup, J. 2007; 31 (3-4): 665-676
  • Long-wavelength analogue of PRODAN: Synthesis and properties of Anthradan, a fluorophore with a 2,6-donor-acceptor anthracene structure JOURNAL OF ORGANIC CHEMISTRY Lu, Z., Lord, S. J., Wang, H., Moerner, W. E., Twieg, R. J. 2006; 71 (26): 9651-9657

    Abstract

    We have synthesized the environment-sensitive fluorophores 2-cyano-6-dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene (Anthradan) starting from 2,6-diaminoanthraquinone. Anthradan is the benzologue of the well-known family of naphthalene 2-propionyl-6-dimethylaminonaphthalene (PRODAN) fluorophores. The additional spectral red shift of the anthracene avoids the autofluorescence of many biological systems and provides for more favorable excitation wavelengths for fluorescence applications. Furthermore, Anthradan exhibits polarity-sensitive emission comparable to that of PRODAN and displays high quantum yields in a range of solvents. Single molecules of these anthracene-containing fluorophores have been imaged in polymer hosts as a proof-of-principle.

    View details for DOI 10.1021/jo0616660

    View details for Web of Science ID 000242845500012

    View details for PubMedID 17168582

  • Single-molecule nanoprobes explore defects in spin-grown crystals JOURNAL OF PHYSICAL CHEMISTRY B Werley, C. A., Moerner, W. E. 2006; 110 (38): 18939-18944

    Abstract

    Thin, platelike single crystals of p-terphenyl (PT) doped with terrylene impurity molecules can be prepared by spin-coating from solution. Strikingly, individual terrylene molecules can be observed traveling inside the crystal over distances of several micrometers by using single-molecule fluorescence imaging at room temperature. Analysis of the motion by single-particle tracking and correlation methods indicates that the molecules act as nanoprobes by exploring long, thin crack-like defects with correlated orientations, defects that can be difficult to observe by other means. Apparently, the regions accessible to the moving molecules are in the interior of the crystal and hence are partially protected from oxidation. In addition to the traveling molecules, which photobleach in times on the order of 32 s under continuous irradiation at 2 kW/cm2, two other spatially fixed populations are observed: one with transition dipole oriented along the c-axis of the crystal with a characteristic photobleaching time greater than 32 h, and one with a characteristic photobleaching time of 18 min.

    View details for DOI 10.1021/jp057570b

    View details for Web of Science ID 000240654900027

    View details for PubMedID 16986887

  • Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, S. Y., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. 2006; 103 (29): 10929-10934

    Abstract

    The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

    View details for DOI 10.1073/pnas.0604503103

    View details for Web of Science ID 000239327200021

    View details for PubMedID 16829583

  • Diffusion of lipid-like single-molecule fluorophores in the cell membrane JOURNAL OF PHYSICAL CHEMISTRY B Nishimura, S. Y., Lord, S. J., Klein, L. O., Willets, K. A., He, M., Lu, Z. K., TWIEG, R. J., Moerner, W. E. 2006; 110 (15): 8151-8157

    Abstract

    The dicyanomethylenedihydrofuran (DCDHF) class of single-molecule fluorophores contains an amine donor and a dicyanomethylenedihydrofuran acceptor linked by a conjugated unit (benzene, naphthalene, or styrene). Molecules in this class have a number of useful properties in addition to those usually required for single-molecule studies (such as high fluorescence quantum yield and photostability), including second-order optical nonlinearity, large ground-state dipole moment, and sensitivity to local environment. Moreover, most DCDHF molecules have amphiphilic structures, with a polar dicyanomethylenedihydrofuran headgroup and nonpolar hydrocarbon tails on the amine or furan ring, and can be used as fluorescent lipid analogues for live cell imaging. Here we demonstrate that individual molecules of several different DCDHF lipid analogues can be observed diffusing in the plasma membrane of Chinese hamster ovary cells. The photophysical and diffusive behaviors of the DCDHF lipid analogues in membranes are described and are found to be competitive with the well-known lipid probe N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine.

    View details for DOI 10.1021/jp0574145

    View details for Web of Science ID 000236992100072

    View details for PubMedID 16610918

  • Suppressing Brownian motion of individual biomolecules in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Moerner, W. E. 2006; 103 (12): 4362-4365

    Abstract

    Single biomolecules in free solution have long been of interest for detailed study by optical methods, but Brownian motion prevents the observation of one single molecule for extended periods. We have used an anti-Brownian electrokinetic (ABEL) trap to trap individual protein molecules in free solution, under ambient conditions, without requiring any attachment to beads or surfaces. We also demonstrate trapping and manipulation of single virus particles, lipid vesicles, and fluorescent semiconductor nanocrystals.

    View details for DOI 10.1073/pnas.0509976103

    View details for Web of Science ID 000236362600009

    View details for PubMedID 16537418

  • Toward nanometer-scale optical photolithography: Utilizing the near-field of bowtie optical nanoantennas NANO LETTERS Sundaramurthy, A., Schuck, P. J., Conley, N. R., Fromm, D. P., Kino, G. S., Moerner, W. E. 2006; 6 (3): 355-360

    Abstract

    Optically resonant metallic bowtie nanoantennas are utilized as fabrication tools for the first time, resulting in the production of polymer resist nanostructures <30 nm in diameter at record low incident multiphoton energy densities. The nanofabrication is accomplished via nonlinear photopolymerization, which is initiated by the enhanced, confined optical fields surrounding the nanoantenna. The position, size, and shape of the resist nanostructures directly correlate with rigorous finite-difference time-domain computations of the field distribution, providing a nanometer-scale measurement of the actual field confinement offered by single optical nanoantennas. In addition, the size of the photoresist regions yields strong upper bounds on photoacid diffusion and resist resolution in SU-8, demonstrating a technique that can be generalized to the study of many current and yet-to-be-developed photoresist systems.

    View details for DOI 10.1021/nl052322c

    View details for Web of Science ID 000236049800004

    View details for PubMedID 16522022

  • Exploring the chemical enhancement for surface-enhanced Raman scattering with Au bowtie nanoantennas JOURNAL OF CHEMICAL PHYSICS Fromm, D. P., Sundaramurthy, A., Kinkhabwala, A., Schuck, P. J., Kino, G. S., Moerner, W. E. 2006; 124 (6)

    Abstract

    Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >10(7), much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules.

    View details for DOI 10.1063/1.2167649

    View details for Web of Science ID 000235309400001

    View details for PubMedID 16483189

  • Cholesterol depletion induces solid-like regions in the plasma membrane BIOPHYSICAL JOURNAL Nishimura, S. Y., Vrljic, M., Klein, L. O., McConnell, H. M., Moerner, W. E. 2006; 90 (3): 927-938

    Abstract

    Glycosylphosphatidylinositol-linked and transmembrane major histocompatibility complex (MHC) class II I-E(k) proteins, as well as N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Tritc-DHPE), are used as probes to determine the effect of cholesterol concentration on the organization of the plasma membrane at temperatures in the range 22 degrees C-42 degrees C. Cholesterol depletion caused a decrease in the diffusion coefficients for the MHC II proteins and also for a slow fraction of the Tritc-DHPE population. At 37 degrees C, reduction of the total cell cholesterol concentration results in a smaller suppression of the translational diffusion for I-E(k) proteins (twofold) than was observed in earlier work at 22 degrees C (five sevenfold) Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Biophys. J. 88:334-347. At 37 degrees C, the diffusion of both I-E(k) proteins is Brownian (0.9 < alpha-parameter < 1.1). More than 99% of the protein population diffuses homogeneously when imaged at 65 frames per s. As the temperature is raised from 22 degrees C to 42 degrees C, a change in activation energy is seen at approximately 35 degrees C in the Arrhenius plots. Cytoskeletal effects appear to be minimal. These results are consistent with a previously described model of solid-like domain formation in the plasma membrane.

    View details for DOI 10.1529/biophysj.105.070524

    View details for Web of Science ID 000234586200022

    View details for PubMedID 16272447

  • Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy JOURNAL OF PHYSICAL CHEMISTRY B Kim, S. Y., Semyonov, A. N., TWIEG, R. J., Horwich, A. L., Frydman, J., Moerner, W. E. 2005; 109 (51): 24517-24525

    Abstract

    Hydrophobic interactions play a major role in binding non-native substrate proteins in the central cavity of the bacterial chaperonin GroEL. The sequence of local conformational changes by which GroEL and its cofactor GroES assist protein folding can be explored using the polarity-sensitive fluorescence probe Nile Red. A specific single-cysteine mutant of GroEL (Cys261), whose cysteine is located inside the central cavity at the apical region of the protein, was covalently labeled with synthetically prepared Nile Red maleimide (NR). Bulk fluorescence spectra of Cys261-NR were measured to examine the effects of binding of the stringent substrate, malate dehydrogenase (MDH), GroES, and nucleotide on the local environment of the probe. After binding denatured substrate, the fluorescence intensity increased by 32 +/- 7%, suggesting enhanced hydrophobicity at the position of the label. On the other hand, in the presence of ATP, the fluorescence intensity decreased by 13 +/- 3%, implying increased local polarity. To explore the sequence of local polarity changes, substrate, GroES, and various nucleotides were added in different orders; the resulting changes in emission intensity provide insight into the sequence of conformational changes occurring during GroEL-mediated protein folding.

    View details for DOI 10.1021/jp0534232

    View details for Web of Science ID 000234259900043

    View details for PubMedID 16375456

  • A novel fluorophore for two-photon-excited single-molecule fluorescence CHEMICAL PHYSICS Schuck, P. J., Willets, K. A., Fromm, D. P., TWIEG, R. J., Moerner, W. E. 2005; 318 (1-2): 7-11
  • Distinct constrictive processes, separated in time and space, divide Caulobacter inner and outer membranes JOURNAL OF BACTERIOLOGY Judd, E. M., Comolli, L. R., Chen, J. C., Downing, K. H., Moerner, W. E., McAdams, H. H. 2005; 187 (20): 6874-6882

    Abstract

    Cryoelectron microscope tomography (cryoEM) and a fluorescence loss in photobleaching (FLIP) assay were used to characterize progression of the terminal stages of Caulobacter crescentus cell division. Tomographic cryoEM images of the cell division site show separate constrictive processes closing first the inner membrane (IM) and then the outer membrane (OM) in a manner distinctly different from that of septum-forming bacteria. FLIP experiments had previously shown cytoplasmic compartmentalization (when cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments) occurring 18 min before daughter cell separation in a 135-min cell cycle so the two constrictive processes are separated in both time and space. In the very latest stages of both IM and OM constriction, short membrane tether structures are observed. The smallest observed pre-fission tethers were 60 nm in diameter for both the inner and outer membranes. Here, we also used FLIP experiments to show that both membrane-bound and periplasmic fluorescent proteins diffuse freely through the FtsZ ring during most of the constriction procession.

    View details for DOI 10.1128/JB.187.20.6874-6882.2005

    View details for Web of Science ID 000232509500003

    View details for PubMedID 16199556

  • Field enhancement and gap-dependent resonance in a system of two opposing tip-to-tip Au nanotriangles PHYSICAL REVIEW B Sundaramurthy, A., Crozier, K. B., Kino, G. S., Fromm, D. P., Schuck, P. J., Moerner, W. E. 2005; 72 (16)
  • Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature JOURNAL OF PHYSICAL CHEMISTRY B Mauring, K., Deich, J., Rosell, F. I., McAnaney, T. B., Moerner, W. E., Boxer, S. G. 2005; 109 (26): 12976-12981

    Abstract

    Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.

    View details for DOI 10.1021/jp0448595

    View details for Web of Science ID 000230224700046

    View details for PubMedID 16852610

  • Nonlinear optical chromophores as nanoscale emitters for single-molecule spectroscopy ACCOUNTS OF CHEMICAL RESEARCH Willets, K. A., Nishimura, S. Y., Schuck, P. J., TWIEG, R. J., Moerner, W. E. 2005; 38 (7): 549-556

    Abstract

    Fluorescence imaging of single molecules at room temperature is a powerful technique for studying complex condensed phase systems and revealing structure and dynamics hidden by ensemble measurements. Successful single-molecule spectroscopic experiments rely upon strong emitters that can be detected at the level of individual copies above the relevant background signals. This Account discusses a class of nonlinear optical chromophores that not only are well-suited for single-molecule imaging but also offer additional beneficial properties such as a significant ground-state dipole moment, moderate hyperpolarizability, and sensitivity to local environment. An overview of the photophysical properties of several members of this class of molecules as well as a mechanism to help understand the environmental sensitivity is presented. Some preliminary applications of the chromophores as single-molecule reporters in cellular and polymer systems are discussed, along with detection of the emitters by two-photon fluorescence.

    View details for DOI 10.1021/ar0401294

    View details for Web of Science ID 000230688000007

    View details for PubMedID 16028889

  • Monolithically integrated semiconductor fluorescence sensor for microfluidic applications SENSORS AND ACTUATORS B-CHEMICAL Thrush, E., Levi, O., Cook, L. J., Deich, J., Kurtz, A., Smith, S. J., Moerner, W. E., Harris, J. S. 2005; 105 (2): 393-399
  • Soliton-induced waveguides in an organic photorefractive glass OPTICS LETTERS Asaro, M., Sheldon, M., Chen, Z. G., Ostroverkhova, O., Moerner, W. E. 2005; 30 (5): 519-521

    Abstract

    We demonstrate optical waveguiding of a probe beam at 980 nm by a soliton beam at 780 nm in an organic photorefractive monolithic glass. Both planar and circular waveguides induced by one- and two-dimensional spatial solitons formed as a result of orientationally enhanced photorefractive nonlinearity are produced in the organic glass. Possibilities for increasing the speed of waveguide formation are discussed.

    View details for Web of Science ID 000227371800023

    View details for PubMedID 15789722

  • Method for trapping and manipulating nanoscale objects in solution APPLIED PHYSICS LETTERS Cohen, A. E., Moerner, W. E. 2005; 86 (9)

    View details for DOI 10.1063/1.1872220

    View details for Web of Science ID 000228991600060

  • Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas PHYSICAL REVIEW LETTERS Schuck, P. J., Fromm, D. P., Sundaramurthy, A., Kino, G. S., Moerner, W. E. 2005; 94 (1)

    Abstract

    Metallic bowtie nanoantennas should provide optical fields that are confined to spatial scales far below the diffraction limit. To improve the mismatch between optical wavelengths and nanoscale objects, we have lithographically fabricated Au bowties with lengths approximately 75 nm and gaps of tens of nm. Using two-photon-excited photoluminescence of Au, the local intensity enhancement factor relative to that for the incident diffraction-limited beam has been experimentally determined for the first time. Enhancements >10(3) occur for 20 nm gap bowties, in good agreement with theoretical simulations.

    View details for DOI 10.1103/PhysRevLett.94.017402

    View details for Web of Science ID 000226308000082

    View details for PubMedID 15698131

  • Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane BIOPHYSICAL JOURNAL Vrljic, M., Nishimura, S. Y., Moerner, W. E., McConnell, H. M. 2005; 88 (1): 334-347

    Abstract

    Glycosylphosphatidylinositol (GPI)-linked and native major histocompatibility complex class II I-E(k) were used as probes to determine the effect of varying cholesterol concentration on the mobility of proteins in the plasma membrane. These proteins were imaged in Chinese hamster ovary cells using single-molecule fluorescence microscopy. Observed diffusion coefficients of both native and GPI-linked I-E(k) proteins were found to depend on cholesterol concentration. As the cholesterol concentration decreases the diffusion coefficients decrease by up to a factor of 7 for native and 5 for GPI-linked I-E(k). At low cholesterol concentrations, after sphingomyelinase treatment, the diffusion coefficients are reduced by up to a factor of 60 for native and 190 for GPI-linked I-E(k). The effect is reversible on cholesterol reintroduction. Diffusion at all studied cholesterol concentrations, for both proteins, appears to be predominantly Brownian for time lags up to 2.5 s when imaged at 10 Hz. A decrease in diffusion coefficients is observed for other membrane proteins and lipid probes, DiIC12 and DiIC18. Fluorescence recovery after photobleaching measurements shows that the fraction of immobile lipid probe increases from 8 to approximately 40% after cholesterol extraction. These results are consistent with the previous work on cholesterol-phospholipid interactions. That is, cholesterol extraction destroys liquid cholesterol-phospholipid complexes, leaving solid-like high melting phospholipid domains that inhibit the lateral diffusion of membrane components.

    View details for Web of Science ID 000226090900031

    View details for PubMedID 15516525

  • Visualization of the movement of single histidine kinase molecules in live Caulobacter cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deich, J., Judd, E. M., McAdams, H. H., Moerner, W. E. 2004; 101 (45): 15921-15926

    Abstract

    The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.

    View details for DOI 10.1073/pnas.0404200101

    View details for Web of Science ID 000225196800020

    View details for PubMedID 15522969

  • Spectral analysis of strongly enhanced visible light transmission through single C-shaped nanoapertures APPLIED PHYSICS LETTERS Matteo, J. A., Fromm, D. P., Yuen, Y., Schuck, P. J., Moerner, W. E., Hesselink, L. 2004; 85 (4): 648-650

    View details for DOI 10.1063/1.1774270

    View details for Web of Science ID 000222855400046

  • Experimental and theoretical investigations of environmentally sensitive single-molecule fluorophores JOURNAL OF PHYSICAL CHEMISTRY B Willets, K. A., Callis, P. R., Moerner, W. E. 2004; 108 (29): 10465-10473

    View details for DOI 10.1021/jp049684d

    View details for Web of Science ID 000222763000031

  • Organic photorefractives: Mechanisms, materials, and applications CHEMICAL REVIEWS Ostroverkhova, O., Moerner, W. E. 2004; 104 (7): 3267-3314

    View details for DOI 10.1021/cr960055c

    View details for Web of Science ID 000222769100003

    View details for PubMedID 15250742

  • Gap-dependent optical coupling of single "Bowtie" nanoantennas resonant in the visible NANO LETTERS Fromm, D. P., Sundaramurthy, A., Schuck, P. J., Kino, G., Moerner, W. E. 2004; 4 (5): 957-961

    View details for DOI 10.1021/nl049951r

    View details for Web of Science ID 000221410000039

  • Integrated semiconductor vertical-cavity surface-emitting lasers and PIN photodetectors for biomedical fluorescence sensing IEEE JOURNAL OF QUANTUM ELECTRONICS Thrush, E., Levi, O., Ha, W., Carey, G., Cook, L. J., Deich, J., Smith, S. J., Moerner, W. E., Harris, J. S. 2004; 40 (5): 491-498
  • Single-molecule fluorescence spectroscopy and microscopy of biomolecular motors ANNUAL REVIEW OF PHYSICAL CHEMISTRY Peterman, E. J., Sosa, H., Moerner, W. E. 2004; 55: 79-96

    Abstract

    The methods of single-molecule fluorescence spectroscopy and microscopy have been recently utilized to explore the mechanism of action of several members of the kinesin and myosin biomolecular motor protein families. Whereas ensemble averaging is removed in single-molecule studies, heterogeneity in the behavior of individual motors can be directly observed, without synchronization. Observation of translocation by individual copies of motor proteins allows analysis of step size, rate, pausing, and other statistical properties of the process. Polarization microscopy as a function of nucleotide state has been particularly useful in revealing new and highly rotationally mobile forms of particular motors. These experiments complement X-ray and biochemical studies and provide a detailed view into the local dynamical behavior of motor proteins.

    View details for DOI 10.1146/annurev.physchem.55.091602.094340

    View details for Web of Science ID 000222766400004

    View details for PubMedID 15117248

  • Laser background characterization in a monolithically integrated biofluorescence sensor ADVANCED BIOMEDICAL AND CLINICAL DIAGNOSTIC SYSTEMS II Thrush, E., Levi, O., Cook, L. J., Deich, J., Smith, S. J., Moerner, W. E., Harris, J. S. 2004; 5318: 59-65

    View details for DOI 10.1117/12.525133

    View details for Web of Science ID 000223125300007

  • Self-trapping of light in an organic photorefractive glass OPTICS LETTERS Chen, Z. G., Asaro, M., Ostroverkhova, O., Moerner, W. E. 2003; 28 (24): 2509-2511

    Abstract

    We report the first observation, to our knowledge, of self-trapping of light as well as optically induced focusing-to-defocusing switching in an organic photorefractive glass, owing to the orientationally enhanced photorefractive nonlinearity of the material.

    View details for Web of Science ID 000187075300025

    View details for PubMedID 14690130

  • Methods of single-molecule fluorescence spectroscopy and microscopy REVIEW OF SCIENTIFIC INSTRUMENTS Moerner, W. E., Fromm, D. P. 2003; 74 (8): 3597-3619

    View details for DOI 10.1063/1.1589587

    View details for Web of Science ID 000184346600001

  • Role of temperature in controlling performance of photorefractive organic glasses CHEMPHYSCHEM Ostroverkhova, O., He, M., TWIEG, R. J., Moerner, W. E. 2003; 4 (7): 732-744

    Abstract

    We present a detailed temperature-dependence study of dielectric, birefringent, conductive, and photorefractive (PR) properties of high-performance low-molecular weight organic glasses that contain 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) derivatives. DCDHF organic glasses sensitized with C60 exhibit high two-beam coupling gain coefficients in the red-wavelength region. However, in the best performing DCDHF glasses at room temperature the PR dynamics are limited by slow molecular reorientation in the electric field. While orientational and, therefore, PR speed can be significantly improved by increasing the temperature above the glass-transition temperature of the material, the steady-state performance may worsen. Comprehensive study of the temperature dependence of various processes, which contribute to the PR effect in DCDHF glasses, clarifies the limiting factors and allows for optimization of the overall PR performance.

    View details for DOI 10.1002/cphc.200200633

    View details for Web of Science ID 000184243600009

    View details for PubMedID 12901305

  • Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Judd, E. M., Ryan, K. R., Moerner, W. E., Shapiro, L., McAdams, H. H. 2003; 100 (14): 8235-8240

    Abstract

    Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

    View details for DOI 10.1073/pnas.1433105100

    View details for Web of Science ID 000184222500037

    View details for PubMedID 12824468

  • High-performance photorefractive organic glass with near-infrared sensitivity APPLIED PHYSICS LETTERS Ostroverkhova, O., Moerner, W. E., He, M., TWIEG, R. J. 2003; 82 (21): 3602-3604

    View details for DOI 10.1063/1.1577214

    View details for Web of Science ID 000182993700006

  • Photorefractive properties of poly(siloxane)-triarylamine-based composites for high-speed applications JOURNAL OF PHYSICAL CHEMISTRY B Wright, D., Gubler, U., Moerner, W. E., DeClue, M. S., Siegel, J. S. 2003; 107 (20): 4732-4737

    View details for DOI 10.1021/jp027456i

    View details for Web of Science ID 000182932900009

  • Synthesis and photorefractive properties of multifunctional glasses CHEMISTRY OF MATERIALS He, M., TWIEG, R. J., Gubler, U., Wright, D., Moerner, W. E. 2003; 15 (5): 1156-1164

    View details for DOI 10.1021/cm020702i

    View details for Web of Science ID 000181441100018

  • Novel fluorophores for single-molecule imaging JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Willets, K. A., Ostroverkhova, O., He, M., TWIEG, R. J., Moerner, W. E. 2003; 125 (5): 1174-1175

    Abstract

    Nonlinear optical chromophores based on dicyanodihydrofuran acceptors paired with amine donors have been found to exhibit sufficiently large fluorescence quantum yields and stability to enable single-molecule detection in polymeric hosts. To illustrate the breadth of this class, six fluorophores are presented, spanning the emission range from 505 to 646 nm. In contrast to conventional single-molecule fluorophores, the new molecules feature sensitivity to local rigidity, large ground-state dipole moments, and large polarizability anisotropies, properties that can be used to design new reporter experiments at the single-molecule level.

    View details for DOI 10.1021/ja029100q

    View details for Web of Science ID 000180713000033

    View details for PubMedID 12553812

  • Novel fluorophores for single-molecule imaging NANOCRYSTALS AND ORGANIC AND HYBRID NANOMATERIALS Willets, K. A., Ostroverkhova, O., Hess, S., He, M., TWIEG, R. J., Moerner, W. E. 2003; 5222: 150-157
  • Synthesis and properties of glassy organic multifunctional photorefractive materials OPTICAL MATERIALS He, M., Twieg, R., Gubler, U., Wright, D., Moerner, W. E. 2003; 21 (1-3): 353-357
  • Optically induced focusing-to-defocusing switching and self-trapping of light in a photorefractive organic glass PHOTOREFRACTIVE EFFECTS, MATERIALS, AND DEVICES, PROCEEDINGS VOLUME Chen, Z. G., Asaro, M., Ostroverkbova, O., Moerner, W. E. 2003; 87: 425-429
  • Translational diffusion of individual class II MHC membrane proteins in cells BIOPHYSICAL JOURNAL Vrljic, M., Nishimura, S. Y., Brasselet, S., Moerner, W. E., McConnell, H. M. 2002; 83 (5): 2681-2692

    Abstract

    Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class II MHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells.

    View details for Web of Science ID 000179024500032

    View details for PubMedID 12414700

  • Synthesis of fluorescently labeled polymers and their use in single-molecule imaging MACROMOLECULES Bowden, N. B., Willets, K. A., Moerner, W. E., Waymouth, R. M. 2002; 35 (21): 8122-8125

    View details for DOI 10.1021/ma020780r

    View details for Web of Science ID 000178444400037

  • Recent advances in understanding and development of photorefractive polymers and glasses ADVANCED FUNCTIONAL MATERIALS Ostroverkhova, O., Wright, D., Gubler, U., Moerner, W. E., He, M., Sastre-Santos, A., TWIEG, R. J. 2002; 12 (9): 621-629
  • Monolithic photorefractive organic glasses with large coupling gain and strong beam fanning ADVANCED MATERIALS Gubler, U., He, M., Wright, D., Roh, Y., Twieg, R., Moerner, W. E. 2002; 14 (4): 313-?
  • Organic photorefractive material design strategies NONLINEAR OPTICAL TRANSMISSION PROCESSES AND ORGANIC PHOTOREFRACTIVE MATERIALS Wright, D., Gubler, U., Sadhukhan, S., Moerner, W. E., He, M., Twieg, R., DeClue, M., Siegel, J. 2002; 4462: 125-138
  • Biomolecular applications of single-molecule measurements : Kinetics and dynamics of a single enzyme reaction METHODS FOR ULTRASENSITIVE DETECTION II Paige, M. F., Fromm, D. P., Moerner, W. E. 2002; 4634: 92-103
  • High-performance photorefractive organic glasses: understanding mechanisms and limitations ORGANIC PHOTOREFRACTIVE AND PHOTOSENSITIVE MATERIALS FOR HOLOGRAPHIC APPLICATIONS Ostroverkhova, O., Gubler, U., Wright, D., He, M., TWIEG, R. J., Moerner, W. E. 2002; 4802: 21-32
  • High-performance photorefractive polymer composite with 2-dicyanomethylen-3-cyano-2,5-dihydrofuran chromophore APPLIED PHYSICS LETTERS Wright, D., Gubler, U., Roh, Y., Moerner, W. E., He, M., TWIEG, R. J. 2001; 79 (26): 4274-4276
  • Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules BIOPHYSICAL JOURNAL Peterman, E. J., Sosa, H., Goldstein, L. S., Moerner, W. E. 2001; 81 (5): 2851-2863

    Abstract

    Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.

    View details for Web of Science ID 000171755200037

    View details for PubMedID 11606296

  • ADP-induced rocking of the kinesin motor domain revealed by single-molecule fluorescence polarization microscopy NATURE STRUCTURAL BIOLOGY Sosa, H., Peterman, E. J., Moerner, W. E., Goldstein, L. S. 2001; 8 (6): 540-544

    Abstract

    Kinesin is an ATP-driven molecular motor protein that moves processively along microtubules. Despite considerable research, the detailed mechanism of kinesin motion remains elusive. We applied an enhanced suite of single- and multiple-molecule fluorescence polarization microscopy assays to report the orientation and mobility of kinesin molecules bound to microtubules as a function of nucleotide state. In the presence of analogs of ATP, ADP-Pi or in the absence of nucleotide, the kinesin head maintains a rigid orientation. In the presence of ADP, the motor domain of kinesin, still bound to the microtubule, adopts a previously undescribed, highly mobile state. This state may be general to the chemomechanical cycle of motor proteins; in the case of kinesin, the transition from a highly mobile to a rigid state after ADP release may contribute to the generation of the 8 nm step.

    View details for Web of Science ID 000168924300019

    View details for PubMedID 11373624

  • Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level JOURNAL OF PHYSICAL CHEMISTRY B Lounis, B., Deich, J., Rosell, F. I., Boxer, S. G., Moerner, W. E. 2001; 105 (21): 5048-5054
  • Photon antibunching in single CdSe/ZnS quantum dot fluorescence CHEMICAL PHYSICS LETTERS Lounis, B., Bechtel, H. A., Gerion, D., Alivisatos, P., Moerner, W. E. 2000; 329 (5-6): 399-404
  • Single photons on demand from a single molecule at room temperature NATURE Lounis, B., Moerner, W. E. 2000; 407 (6803): 491-493

    Abstract

    The generation of non-classical states of light is of fundamental scientific and technological interest. For example, 'squeezed' states enable measurements to be performed at lower noise levels than possible using classical light. Deterministic (or triggered) single-photon sources exhibit non-classical behaviour in that they emit, with a high degree of certainty, just one photon at a user-specified time. (In contrast, a classical source such as an attenuated pulsed laser emits photons according to Poisson statistics.) A deterministic source of single photons could find applications in quantum information processing, quantum cryptography and certain quantum computation problems. Here we realize a controllable source of single photons using optical pumping of a single molecule in a solid. Triggered single photons are produced at a high rate, whereas the probability of simultaneous emission of two photons is nearly zero--a useful property for secure quantum cryptography. Our approach is characterized by simplicity, room temperature operation and improved performance compared to other triggered sources of single photons.

    View details for Web of Science ID 000089727400043

    View details for PubMedID 11028995

  • Single-molecule fluorescence resonant energy transfer in calcium concentration dependent cameleon JOURNAL OF PHYSICAL CHEMISTRY B Brasselet, S., Peterman, E. J., Miyawaki, A., Moerner, W. E. 2000; 104 (15): 3676-3682
  • Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwille, P., Kummer, S., Heikal, A. A., Moerner, W. E., Webb, W. W. 2000; 97 (1): 151-156

    Abstract

    Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10(-6)-10(-3)-s time range, by using fluorescence correlation spectroscopy at 10(-8) M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over approximately 6 x 10(2) to 5 x 10(6) W/cm(2). These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of approximately 10(-3). Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-microseconds time range. The independence of these two bright-dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.

    View details for Web of Science ID 000084624500029

    View details for PubMedID 10618386

  • Photorefractive polymers for laser-based ultrasound detection ORGANIC PHOTOREFRACTIVES, PHOTORECEPTORS, AND NANOCOMPOSITES Wright, D., Gubler, U., Klein, M. B., Moerner, W. E. 2000; 4104: 110-117
  • The fluorescence dynamics of single molecules of green fluorescent protein JOURNAL OF PHYSICAL CHEMISTRY A Peterman, E. J., Brasselet, S., Moerner, W. E. 1999; 103 (49): 10553-10560
  • Photorefractive properties of poly(N-vinyl carbazole)-based composites for high-speed applications CHEMISTRY OF MATERIALS Diaz-Garcia, M. A., Wright, D., Casperson, J. D., Smith, B., Glazer, E., Moerner, W. E., Sukhomlinova, L. I., TWIEG, R. J. 1999; 11 (7): 1784-1791
  • Optical methods for exploring dynamics of single copies of green fluorescent protein CYTOMETRY Moerner, W. E., Peterman, E. J., Brasselet, S., Kummer, S., Dickson, R. M. 1999; 36 (3): 232-238

    Abstract

    Single copies of four different phenolate ion mutants of the green fluorescent protein (GFP) exhibit a complex blinking and fluctuating behavior, a phenomenon that is hidden in measurements on large ensembles. Both total internal reflection microscopy and scanning confocal microscopy can be used to study the blinking dynamics, and autocorrelation analysis yields histograms of the correlation times for many individual molecules. While the total internal reflection method can follow several single molecules simultaneously, the confocal method offers higher time resolution at the expense of parallelism. We compare and contrast the two methods in terms of the ability to follow the complex dynamics of this system.

    View details for Web of Science ID 000081024800013

    View details for PubMedID 10404973

  • Homodyne detection of ultrasonic surface displacements using two-wave mixing in photorefractive polymers OPTICS COMMUNICATIONS Klein, M. B., Bacher, G. D., Grunnet-Jepsen, A., Wright, D., Moerner, W. E. 1999; 162 (1-3): 79-84
  • Illuminating single molecules in condensed matter SCIENCE Moerner, W. E., Orrit, M. 1999; 283 (5408): 1670-?

    Abstract

    Efficient collection and detection of fluorescence coupled with careful minimization of background from impurities and Raman scattering now enable routine optical microscopy and study of single molecules in complex condensed matter environments. This ultimate method for unraveling ensemble averages leads to the observation of new effects and to direct measurements of stochastic fluctuations. Experiments at cryogenic temperatures open new directions in molecular spectroscopy, quantum optics, and solid-state dynamics. Room-temperature investigations apply several techniques (polarization microscopy, single-molecule imaging, emission time dependence, energy transfer, lifetime studies, and the like) to a growing array of biophysical problems where new insight may be gained from direct observations of hidden static and dynamic inhomogeneity.

    View details for Web of Science ID 000079102800037

    View details for PubMedID 10073924

  • Design and optimization of chromophores for liquid crystal and photorefractive applications ORGANIC NONLINEAR OPTICAL MATERIALS AND DEVICES TWIEG, R. J., He, M., Sukhomlinova, L., You, F., Moerner, W. E., Diaz-Garcia, M. A., Wright, D., Casperson, J. D., Wortmann, R., Glania, C., Kramer, P., Lukaszuk, K., Matschiner, R., Singer, K. D., Ostoverkhov, V., Petschek, R. 1999; 561: 119-130
  • Homodyne detection of ultrasonic surface displacements using two-wave mixing in photorefractive polymers PROCESS CONTROL AND SENSORS FOR MANUFACTURING II Klein, M. B., Bacher, G. D., Grunnet-Jepsen, A., Wright, D., Moerner, W. E. 1999; 3589: 22-29
  • Simultaneous imaging of individual molecules aligned both parallel and perpendicular to the optic axis PHYSICAL REVIEW LETTERS Dickson, R. M., Norris, D. J., Moerner, W. E. 1998; 81 (24): 5322-5325
  • Synthesis of bifunctional photorefractive polymers with net gain: Design strategy amenable to combinatorial optimization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bratcher, M. S., DeClue, M. S., Grunnet-Jepsen, A., Wright, D., Smith, B. R., Moerner, W. E., Siegel, J. S. 1998; 120 (37): 9680-9681
  • High-speed photorefractive polymer composites APPLIED PHYSICS LETTERS Wright, D., Diaz-Garcia, M. A., Casperson, J. D., DeClue, M., Moerner, W. E., TWIEG, R. J. 1998; 73 (11): 1490-1492
  • Spectroscopic determination of trap density in C-60-sensitized photorefractive polymers CHEMICAL PHYSICS LETTERS Grunnet-Jepsen, A., Wright, D., Smith, B., Bratcher, M. S., DeClue, M. S., Siegel, J. S., Moerner, W. E. 1998; 291 (5-6): 553-561
  • Systematics of two-wave mixing in a photorefractive polymer JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Grunnet-Jepsen, A., Thompson, C. L., Moerner, W. E. 1998; 15 (2): 905-913
  • Amplified scattering in a high-gain photorefractive polymer JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Grunnet-Jepsen, A., Thompson, C. L., TWIEG, R. J., Moerner, W. E. 1998; 15 (2): 901-904
  • Gain enhancement by moving gratings in a photorefractive polymer OPTICS COMMUNICATIONS Grunnet-Jepsen, A., Thompson, C. L., Moerner, W. E. 1998; 145 (1-6): 145-149
  • Probing single molecules in polyacrylamide gels LASER TECHNIQUES FOR CONDENSED-PHASE AND BIOLOGICAL SYSTEMS Kummer, S., Dickson, R. M., Moerner, W. E. 1998; 3273: 165-173
  • Trapping studies on photorefractive polymers XEROGRAPHIC PHOTORECEPTORS AND ORGANIC PHOTOREFRACTIVE MATERIALS IV Wright, D., Grunnet-Jepsen, A., Diaz-Garcia, M. A., Casperson, J. D., Smith, B., Bratcher, M. S., DeClue, M. S., Siegel, J. S., Moerner, W. E., TWIEG, R. J. 1998; 3471: 60-71
  • Single-molecule nanophotonics in solids MATERIALS SCIENCE AND ENGINEERING B-SOLID STATE MATERIALS FOR ADVANCED TECHNOLOGY Moerner, W. E., Dickson, R. M., Norris, D. J. 1997; 48 (1-2): 169-174
  • Spontaneous oscillation and self-pumped phase conjugation in a photorefractive polymer optical amplifier SCIENCE GRUNNETJEPSEN, A., Thompson, C. L., Moerner, W. E. 1997; 277 (5325): 549-552
  • On/off blinking and switching behaviour of single molecules of green fluorescent protein NATURE Dickson, R. M., Cubitt, A. B., Tsien, R. Y., Moerner, W. E. 1997; 388 (6640): 355-358

    Abstract

    Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.

    View details for Web of Science ID A1997XM52800043

    View details for PubMedID 9237752

  • Measurement of the spatial phase shift in high-gain photorefractive materials OPTICS LETTERS GRUNNETJEPSEN, A., Thompson, C. L., Moerner, W. E. 1997; 22 (12): 874-876

    Abstract

    The correct determination of the spatial phase shift ĝ(p) in photorefractive materials is crucial to the proper characterization of novel materials. It is shown that the grating translation techniques commonly used for the measurement of ĝ(p) need to be reevaluated for high-gain materials. Strong energy and phase coupling leads to nonuniform slanted gratings, which result in an apparent dependence of the phase shift of the beam ratio and the optical polarization. A revised theory is presented, and analytical solutions are obtained for the special case of ĝ(p)?pi/2 . Numerical solutions for arbitrary ĝ(p) are in good agreement with measurements in a photorefractive polymer.

    View details for Web of Science ID A1997XE10700010

    View details for PubMedID 18185691

  • High performance photorefractive polymer with improved stability APPLIED PHYSICS LETTERS GRUNNETJEPSEN, A., Thompson, C. L., TWIEG, R. J., Moerner, W. E. 1997; 70 (12): 1515-1517
  • Single-molecule spectroscopy and quantum optics in solids ADVANCES IN ATOMIC, MOLECULAR, AND OPTICAL PHYSICS Moerner, W. E., Dickson, R. M., Norris, D. J. 1997; 38: 193-236
  • Optical limiting in a photorefractive polymer MATERIALS FOR OPTICAL LIMITING II Grunnet-Jepsen, A., Thompson, C. L., Moerner, W. E. 1997; 479: 199-207
  • Recent advances in photorefractive polymer materials NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS X Moerner, W. E., GRUNNETJEPSEN, A., Thompson, C. L., Bratcher, M. S., TWIEG, R. J. 1997; 3147: 84-94
  • Large gain photorefractive polymers XEROGRAPHIC PHOTORECEPTORS AND ORGANICS PHOTOREFRACTIVE MATERIALS II GRUNNETJEPSEN, A., Thompson, C. L., TWIEG, R. J., Belfield, K., Bratcher, M. S., Moerner, W. E. 1997; 3144: 216-226
  • Photorefractive polymers ANNUAL REVIEW OF MATERIALS SCIENCE Moerner, W. E., GRUNNETJEPSEN, A., Thompson, C. L. 1997; 27: 585-623
  • Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels SCIENCE Dickson, R. M., Norris, D. J., Tzeng, Y. L., Moerner, W. E. 1996; 274 (5289): 966-969

    Abstract

    Individual fluorescent molecules and individual singly labeled proteins were observed in the water-filled pores of poly(acrylamide) gels by far-field microscopy. Brownian motion was markedly reduced by the gel framework, thus enabling extended study of single fluorophores in aqueous environments. A highly axially dependent laser field was used both to excite the fluorophores and to image the molecules in three dimensions. Single molecules were followed as they moved within and through the porous gel structure. In contrast to dry polymeric hosts, these water-based gels may form a useful medium for single-molecule studies of biological systems in vitro.

    View details for Web of Science ID A1996VR79200044

    View details for PubMedID 8875935

  • Single molecules solvated in pores of polyacrylamide gels MOLECULAR CRYSTALS AND LIQUID CRYSTALS SCIENCE AND TECHNOLOGY SECTION A-MOLECULAR CRYSTALS AND LIQUID CRYSTALS Dickson, R. M., Norris, D. J., Tzeng, Y. L., Sakowicz, R., Goldstein, L. S., Moerner, W. E. 1996; 291: 31-39
  • Mechanisms of photorefractivity in polymer composites ORGANIC PHOTOREFRACTIVE MATERIALS AND XEROGRAPHIC PHOTORECEPTORS Moerner, W. E., GRUNNETJEPSEN, A., Thompson, C. L., TWIEG, R. J. 1996; 2850: 2-13
  • PHOTOREFRACTIVE POLYMERS BASED ON DUAL-FUNCTION DOPANTS JOURNAL OF PHYSICAL CHEMISTRY SILENCE, S. M., Scott, J. C., Stankus, J. J., Moerner, W. E., Moylan, C. R., Bjorklund, G. C., TWIEG, R. J. 1995; 99 (12): 4096-4105
  • PHOTOREFRACTIVE POLYMERS - A STATUS-REPORT PURE AND APPLIED CHEMISTRY BURLAND, D. M., Bjorklund, G. C., Moerner, W. E., SILENCE, S. M., Stankus, J. J. 1995; 67 (1): 33-38
  • Photorefractivity in new organic polymeric materials XEROGRAPHIC PHOTORECEPTORS AND PHOTOREFRACTIVE POLYMERS Poga, C., BURLAND, D. M., Hanemann, T., Jia, Y., Moylan, C. R., Stankus, J. J., TWIEG, R. J., Moerner, W. E. 1995; 2526: 82-93
  • OPTICAL TRAP ACTIVATION IN A PHOTOREFRACTIVE POLYMER OPTICS LETTERS SILENCE, S. M., Bjorklund, G. C., Moerner, W. E. 1994; 19 (22): 1822-1824

    View details for Web of Science ID A1994PR09400008

    View details for PubMedID 19855665

  • CASCADING OF 2ND-ORDER PROCESSES IN QUADRATIC MOLECULAR MEDIA AT THE ORIGIN OF VERY LARGE CUBIC EFFECTS SYNTHETIC METALS Vidakovic, P., Zyss, J., Kim, D., TORUELLAS, W., Stegeman, G., Moerner, W. E., Twieg, R., Bjorklund, G. 1994; 67 (1-3): 303-307
  • QUASINONDESTRUCTIVE READOUT IN A PHOTOREFRACTIVE POLYMER PHYSICAL REVIEW LETTERS SILENCE, S. M., TWIEG, R. J., Bjorklund, G. C., Moerner, W. E. 1994; 73 (15): 2047-2050
  • ELECTRIC-FIELD-SWITCHABLE STRATIFIED VOLUME HOLOGRAMS IN PHOTOREFRACTIVE POLYMERS OPTICS LETTERS Stankus, J. J., SILENCE, S. M., Moerner, W. E., Bjorklund, G. C. 1994; 19 (18): 1480-1482

    Abstract

    Two- and four-layer stratified volume holograms have been fabricated from the photorefractive polymeric material poly(methyl methacrylate): (1,3-dimethyl-2,2-tetramethylene-5-nitrobenzimidazoline):C(60) and characterized by holographic four-wave mixing experiments. Coherent addition of diffracted fields from the individual layers is observed, leading to a diffraction efficiency that increases with the square of the active layer thickness. Electricfield switching of the diffraction efficiencies of individual layers is demonstrated. The angular selectivity of the diffraction efficiency is also characterized for one, two, and four active layers. The angular width of the peaks narrows with increasing total structure thickness in agreement with theory.

    View details for Web of Science ID A1994PJ26400035

    View details for PubMedID 19855559

  • 2ND-ORDER CASCADING AS THE ORIGIN OF LARGE 3RD-ORDER EFFECTS IN ORGANIC SINGLE-CRYSTAL-CORE FIBERS OPTICS LETTERS Kim, D. Y., Torruellas, W. E., Kang, J., Bosshard, C., Stegeman, G. I., Vidakovic, P., Zyss, J., Moerner, W. E., Twieg, R., Bjorklund, G. 1994; 19 (12): 868-870

    Abstract

    We have identified cascading of second-order nonlinear processes as the origin of previously reported, very large nonlinearities measured by self-phase-modulation experiments in organic single-crystal-core fibers of 4-(N,N-dimethylamino)-3-acetamidonitrobenzene.

    View details for Web of Science ID A1994NQ38600006

    View details for PubMedID 19844471

  • OPTICAL-PROPERTIES OF POLY(N-VINYLCARBAZOLE)-BASED GUEST-HOST PHOTOREFRACTIVE POLYMER SYSTEMS APPLIED OPTICS SILENCE, S. M., Donckers, M. C., Walsh, C. A., BURLAND, D. M., TWIEG, R. J., Moerner, W. E. 1994; 33 (11): 2218-2222

    Abstract

    The photorefractive properties of poly(N-vinylcarbazole) doped with a variety of nonlinear optical chromophores and sensitizing agents are surveyed. Steady-state diffraction efficiencies of greater than 10(-3) and two-beam coupling gain exceeding the absorption loss are found in six materials combinations. The effect of the structure of the nonlinear optical chromophore on the photorefractive properties is discussed.

    View details for Web of Science ID A1994NE30400020

    View details for PubMedID 20885569

  • ELECTRIC FIELD-DEPENDENT NONPHOTOREFRACTIVE GRATINGS IN A NONLINEAR PHOTOCONDUCTING POLYMER APPLIED PHYSICS LETTERS SILENCE, S. M., Donckers, M. C., Walsh, C. A., BURLAND, D. M., Moerner, W. E., TWIEG, R. J. 1994; 64 (6): 712-714
  • ORIENTATIONALLY ENHANCED PHOTOREFRACTIVE EFFECT IN POLYMERS JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Moerner, W. E., SILENCE, S. M., Hache, F., Bjorklund, G. C. 1994; 11 (2): 320-330
  • POLYMERIC PHOTOREFRACTIVE MATERIALS CHEMICAL REVIEWS Moerner, W. E., SILENCE, S. M. 1994; 94 (1): 127-155
  • RECENT PROGRESS IN PHOTOREFRACTIVE POLYMERS - MATERIALS AND STRUCTURES NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS VII Stankus, J. J., SILENCE, S. M., TWIEG, R. J., BURLAND, D. M., Miller, R. D., Scott, J. C., Moerner, W. E., Bjorklund, G. C. 1994; 2285: 204-215
  • POLY(SILANE)-BASED HIGH-MOBILITY PHOTOREFRACTIVE POLYMERS JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS SILENCE, S. M., Scott, J. C., Hache, F., Ginsburg, E. J., JENKNER, P. K., Miller, R. D., TWIEG, R. J., Moerner, W. E. 1993; 10 (12): 2306-2312
  • NET 2-BEAM-COUPLING GAIN IN A POLYMERIC PHOTOREFRACTIVE MATERIAL OPTICS LETTERS Donckers, M. C., SILENCE, S. M., Walsh, C. A., Hache, F., BURLAND, D. M., Moerner, W. E., TWIEG, R. J. 1993; 18 (13): 1044-1046

    View details for Web of Science ID A1993LJ22700004

    View details for PubMedID 19823284

  • OPTICAL SPECTROSCOPY OF SINGLE IMPURITY MOLECULES IN SOLIDS ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Moerner, W. E., Basche, T. 1993; 32 (4): 457-476
  • NONLINEAR-OPTICAL PROPERTIES OF PHOTOREFRACTIVE POLYMERS NONLINEAR OPTICAL PROPERTIES OF ADVANCED MATERIALS SILENCE, S. M., Hache, F., Donckers, M., Walsh, C. A., BURLAND, D. M., Bjorklund, G. C., TWIEG, R. J., Moerner, W. E. 1993; 1852: 253-265
  • LIGHTWAVE TRANSMISSION OF MULTIPLE TELEVISION SIGNALS USING AN ORGANIC POLYMER ELECTROOPTIC PHASE MODULATOR NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS VI SMITH, B. A., Jurich, M., Moerner, W. E., Volksen, W., Best, M. E., Fleming, W., Swalen, J. D., Bjorklund, G. C. 1993; 2025: 499-506
  • C-60 SENSITIZATION OF A PHOTOREFRACTIVE POLYMER APPLIED PHYSICS LETTERS SILENCE, S. M., Walsh, C. A., Scott, J. C., Moerner, W. E. 1992; 61 (25): 2967-2969
  • PHOTOCONDUCTIVITY STUDIES OF PHOTOREFRACTIVE POLYMERS JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Scott, J. C., PAUTMEIER, L. T., Moerner, W. E. 1992; 9 (11): 2059-2064
  • PHOTON ANTIBUNCHING IN THE FLUORESCENCE OF A SINGLE DYE MOLECULE TRAPPED IN A SOLID PHYSICAL REVIEW LETTERS Basche, T., Moerner, W. E., Orrit, M., TALON, H. 1992; 69 (10): 1516-1519
  • SUBSECOND GRATING GROWTH IN A PHOTOREFRACTIVE POLYMER OPTICS LETTERS SILENCE, S. M., Walsh, C. A., Scott, J. C., MATRAY, T. J., TWIEG, R. J., Hache, F., Bjorklund, G. C., Moerner, W. E. 1992; 17 (16): 1107-1109

    Abstract

    We survey the dynamics of the photorefractive effect in a methyl methacrylate copolymer with the nonlinear chromophore p-nitroaniline in a pendant side group doped with a charge-transport agent, diethylaminobenzaldehyde diphenylhydrazone, a material that represents a new class of photorefractive polymer. The grating growth times are several orders of magnitude smaller than that for the previous epoxy-based photorefractive polymers and fall below 1 s at the highest intensities used. Grating competition and revelation effects suggest that charge carriers other than photogenerated holes are mobile. A sublinear dependence of growth rate on writing intensity implies that shallow traps may also be present.

    View details for Web of Science ID A1992JG55500003

    View details for PubMedID 19794733

  • SINGLE MOLECULE SPECTRAL DIFFUSION IN A SOLID DETECTED VIA FLUORESCENCE SPECTROSCOPY JOURNAL OF LUMINESCENCE Ambrose, W. P., Basche, T., Moerner, W. E. 1992; 53 (1-6): 62-67
  • OPTICAL-SPECTRA AND KINETICS OF SINGLE IMPURITY MOLECULES IN A POLYMER - SPECTRAL DIFFUSION AND PERSISTENT SPECTRAL HOLE BURNING JOURNAL OF THE OPTICAL SOCIETY OF AMERICA B-OPTICAL PHYSICS Basche, T., Ambrose, W. P., Moerner, W. E. 1992; 9 (5): 829-836
  • OPTICAL MODIFICATION OF A SINGLE IMPURITY MOLECULE IN A SOLID NATURE Basche, T., Moerner, W. E. 1992; 355 (6358): 335-337
  • PHOTOCONDUCTIVITY OF PHOTOREFRACTIVE POLYMERS MACROMOLECULAR HOST-GUEST COMPLEXES : OPTICAL, OPTOELECTRONIC, AND PHOTOREFRACTIVE PROPERTIES AND APPLICATIONS Scott, J. C., PAUTMEIER, L. T., Moerner, W. E., Walsh, C. A., SILENCE, S. M., MATRAY, T. J., TWIEG, R. J. 1992; 277: 135-144
  • OPTICAL WAVE-GUIDING IN POLED NLO POLYMERS MATERIALS FOR OPTICAL INFORMATION PROCESSING Swalen, J. D., Fleming, W., Jurich, M., Moerner, W. E., SMITH, B. A., Herminghaus, S., Bjorklund, G. C. 1992; 228: 101-110
  • NONLINEAR OPTICAL-PROPERTIES OF ORGANIC PHOTOREFRACTIVE POLYMERS MACROMOLECULAR HOST-GUEST COMPLEXES : OPTICAL, OPTOELECTRONIC, AND PHOTOREFRACTIVE PROPERTIES AND APPLICATIONS Moerner, W. E., Walsh, C. A., SILENCE, S. M., TWIEG, R. J., MATRAY, T. J., Scott, J. C., Lee, V. Y., Miller, R. D., Hache, F., BURLAND, D. M., Bjorklund, G. C. 1992; 277: 121-133
  • NLO POLYMERIC WAVE-GUIDE ELECTROOPTIC PHASE MODULATOR NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS V Swalen, J. D., Bjorklund, G. C., Fleming, W., Hung, R., Jurich, M., Lee, V. Y., Miller, R. D., Moerner, W. E., MORICHERE, D. Y., Skumanich, A., SMITH, B. A. 1992; 1775: 369-378
  • DETECTION AND SPECTROSCOPY OF SINGLE PENTACENE MOLECULES IN A PARA-TERPHENYL CRYSTAL BY MEANS OF FLUORESCENCE EXCITATION JOURNAL OF CHEMICAL PHYSICS Ambrose, W. P., Basche, T., Moerner, W. E. 1991; 95 (10): 7150-7163
  • OBSERVATION OF THE PHOTOREFRACTIVE EFFECT IN A POLYMER PHYSICAL REVIEW LETTERS Ducharme, S., Scott, J. C., TWIEG, R. J., Moerner, W. E. 1991; 66 (14): 1846-1849
  • APPLICATIONS OF ORGANIC 2ND-ORDER NONLINEAR OPTICAL-MATERIALS ACS SYMPOSIUM SERIES Bjorklund, G. C., Ducharme, S., Fleming, W., JUNGBAUER, D., Moerner, W. E., Swalen, J. D., TWIEG, R. J., WILLSON, C. G., Yoon, D. Y. 1991; 455: 216-225
  • POLED EPOXY POLYMERS FOR OPTOELECTRONICS ORGANIC MOLECULES FOR NONLINEAR OPTICS AND PHOTONICS Swalen, J. D., Bjorklund, G. C., Fleming, W., Herminghaus, S., JUNGBAUER, D., Jurich, M., Moerner, W. E., Reck, B., SMITH, B. A., Twieg, R., WILLSON, C. G., Zentel, R. 1991; 194: 433-445
  • FINDING A SINGLE MOLECULE IN A HAYSTACK - LASER SPECTROSCOPY OF SOLIDS FROM SQUARE-ROOT-N TO N=1 OPTICAL METHODS FOR ULTRASENSITIVE DETECTION AND ANALYSIS : TECHNIQUES AND APPLICATIONS Moerner, W. E., Ambrose, W. P. 1991; 1435: 244-251
  • PHOTOREFRACTIVITY IN DOPED NONLINEAR ORGANIC POLYMERS NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS IV Moerner, W. E., Walsh, C., Scott, J. C., Ducharme, S., BURLAND, D. M., Bjorklund, G. C., TWIEG, R. J. 1991; 1560: 278-289
  • INTRACAVITY FREQUENCY DOUBLING OF A ND-YAG LASER WITH AN ORGANIC NONLINEAR OPTICAL-CRYSTAL APPLIED PHYSICS LETTERS Ducharme, S., Risk, W. P., Moerner, W. E., Lee, V. Y., TWIEG, R. J., Bjorklund, G. C. 1990; 57 (6): 537-539
  • OPTICAL-DETECTION AND PROBING OF SINGLE DOPANT MOLECULES OF PENTACENE IN A P-TERPHENYL HOST CRYSTAL BY MEANS OF ABSORPTION-SPECTROSCOPY JOURNAL OF PHYSICAL CHEMISTRY Kador, L., HORNE, D. E., Moerner, W. E. 1990; 94 (4): 1237-1248
  • ORGANIC NONLINEAR OPTICAL-MATERIALS AND THEIR DEVICE APPLICATIONS FOR FREQUENCY DOUBLING, MODULATION, AND SWITCHING NONLINEAR OPTICAL PROPERTIES OF ORGANIC MATERIALS III Swalen, J. D., Bjorklund, G. C., Ducharme, S., Fleming, W., Herminghaus, S., JUNGBAUER, D., Moerner, W. E., SMITH, B. A., Twieg, R., Yoon, D., Willson, G. 1990; 1337: 2-11
  • ULTRASENSITIVE LASER SPECTROSCOPY IN SOLIDS - SINGLE-MOLECULE DETECTION MOLECULAR CRYSTALS AND LIQUID CRYSTALS Moerner, W. E., Kador, L., Ambrose, W. P. 1990; 183: 47-57
  • FINDING A SINGLE MOLECULE IN A HAYSTACK - OPTICAL-DETECTION AND SPECTROSCOPY OF SINGLE ABSORBERS IN SOLIDS ANALYTICAL CHEMISTRY Moerner, W. E., Kador, L. 1989; 61 (21): A1217-A1223
  • OPTICAL-DETECTION AND SPECTROSCOPY OF SINGLE MOLECULES IN A SOLID PHYSICAL REVIEW LETTERS Moerner, W. E., Kador, L. 1989; 62 (21): 2535-2538

Conference Proceedings


  • Enzymatic Activation of Nitro-Aryl Fluorogens in Live Cells for Turnover Activated Localization Microscopy Lee, M., Williams, J., Twieg, R., Rao, J., Moerner, W. WILEY-BLACKWELL. 2012: 127-127
  • Photoactivatable azido push-pull fluorophores for single-molecule imaging in and out of cells Lord, S. J., Conley, N. R., Lee, H. D., Lee, M. K., Liu, N., Samuel, R., Twieg, R. J., Moerner, W. E. AMER CHEMICAL SOC. 2010
  • Superresolution imaging of protein superstructures in live Caulobacter crescentus cells with EYFP Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L., Moerner, W. E. AMER CHEMICAL SOC. 2009
  • Imaging beyond the diffraction limit in cells using single-molecule active control Moerner, W. E., Biteen, J., Conley, N. R., Lee, H., Lord, S. J., Thompson, M. A., Shapiro, L., Liu, N., Samuel, R., Twieg, R. J. AMER CHEMICAL SOC. 2009: 555-555
  • Dicyanomethylenedihydrofuran (DCDHF) single molecule fluorophores for cellular applications Wang, H., Lu, Z., Lord, S. J., Willets, K. A., Moerner, W. E., Twieg, R. J. AMER CHEMICAL SOC. 2007
  • COLL 60-Single-molecule fluorescence tracking probes membrane dynamics Moerner, W. E., Hwang, H., Kim, S. Y., Kinkhabwala, A., Nishimura, S. AMER CHEMICAL SOC. 2006
  • PHYS 496-Lateral movements of single poly(arginine) peptides on the CHO plasma membrane before cellular entry Hwang, H., Kim, S. Y., Goun, E., Jones, L. R., Wender, P. A., Semyonov, A. N., Twieg, R. J., Moerner, W. E. AMER CHEMICAL SOC. 2006
  • PHYS 665-An improved single-molecule molecular beacon utilizing H-dimer excitonic behavior Conley, N. R., Kurtz, A. H., Wang, H., Twieg, R. J., Moerner, W. E. AMER CHEMICAL SOC. 2006
  • PHYS 484-DCDHF dyes can probe environmental changes by protein binding in GroEL Kim, S. Y., Cui, J., Lu, Z., Semyonov, A. N., Twieg, R. W., Moerner, W. E. AMER CHEMICAL SOC. 2006
  • PHYS 488-DCDHF photophysics: Designing new single-molecule fluorophores for cellular imaging Lord, S. J., Lu, Z., Wang, H., Alyono, J., Liu, N., Weber, R., Twieg, R. J., Moerner, W. E. AMER CHEMICAL SOC. 2006
  • PHYS 252-Single-molecule fluorescence imaging reports on biomolecular dynamics Moerner, W. E., Cohen, A., Conley, N. R., Kim, S. Y., Kinkhabwala, A., Koenig, M., Kurtz, A. H., Lord, S. J., Lu, Z., Wang, H., Twieg, R. J. AMER CHEMICAL SOC. 2006
  • Synthesis and properties of the highly environment sensitive fluorophores 2-cyano and 2-propionyl-6-dihexylaminoanthracene (ANTHRADAN) Lu, Z., Wang, H., Lord, S. J., Moerner, W. E., Twieg, R. J. AMER CHEMICAL SOC. 2006
  • Single-molecule imaging in Caulobacter crescentus. Paige, M. F., Judd, E., Shapiro, L., Moerner, W. E. AMER CHEMICAL SOC. 2001: U285-U285

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