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W E Moerner

Publications and source records attributed to W E Moerner.

At least 19 recordsLinked to original sources
PubMed2006

Long-wavelength analogue of PRODAN: synthesis and properties of Anthradan, a fluorophore with a 2,6-donor-acceptor anthracene structure.

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.

Anthracenes↗
PubMed2006

Single-molecule nanoprobes explore defects in spin-grown crystals.

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.

Crystallization↗
PubMed2006

Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus.

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.

Actins↗
PubMed2006

Diffusion of lipid-like single-molecule fluorophores in the cell membrane.

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.

Animals↗
PubMed2006

Suppressing Brownian motion of individual biomolecules in solution.

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.

Fluorescent Dyes↗
PubMed2006

Exploring the chemical enhancement for surface-enhanced Raman scattering with Au bowtie nanoantennas.

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.

Absorption↗
PubMed2006

Toward nanometer-scale optical photolithography: utilizing the near-field of bowtie optical nanoantennas.

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.

Color↗
PubMed2005

Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy.

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.

Adenosine Triphosphate↗
PubMed2005

Cholesterol depletion induces solid-like regions in the plasma membrane.

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.

Animals↗
PubMed2005

Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature.

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.

Green Fluorescent Proteins↗
PubMed2005

Soliton-induced waveguides in an organic photorefractive glass.

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.

Journal Article↗
PubMed2005

Improving the mismatch between light and nanoscale objects with gold bowtie nanoantennas.

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.

Journal Article↗
PubMed2005

Nonlinear optical chromophores as nanoscale emitters for single-molecule spectroscopy.

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.

Fluorescent Dyes↗
PubMed2005

Distinct constrictive processes, separated in time and space, divide caulobacter inner and outer membranes.

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.

Bacterial Outer Membrane Proteins↗
PubMed2004

Visualization of the movement of single histidine kinase molecules in live Caulobacter cells.

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.

Bacterial Proteins↗
PubMed2004

Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane.

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.

Actins↗