Biomedical subjects

Erwen Mei

Publications and source records attributed to Erwen Mei.

8 recordsLinked to original sources

PubMed2006

High-resolution optical imaging from trajectory time distributions.

Trajectory time distribution optical microscopy (TTDOM), which records the mean off-times of single molecular fluorescent indicators that light up when they collide with vesicles, is extended to record fluorescence durations or on-times. TTDOM can distinguish shapes of objects that are smaller than the diffraction limited resolution. The fluorescence duration time image can also provide high-resolution information. The effects of the threshold that separates fluorescent bursts from background signals and of two or more probes visiting the vesicles simultaneously have been investigated systematically. New experimental results along with simulations indicate that TTDOM is capable of providing the size and shape of objects and information on probe-vesicle binding.

Dimyristoylphosphatidylcholine↗

PubMed2006

Probing lipid vesicles by bimolecular association and dissociation trajectories of single molecules.

Vesicles prepared by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid molecules having sizes smaller than the diffraction-limited focused laser beam have been used to confine single molecules in the laser focus. The confinement of single molecules in a volume smaller than the focused laser beam leads to a Gaussian distribution of single molecule fluorescence intensity. The interactions of single Nile Red molecules with DMPC and SOPC lipid bilayers were studied by single molecule fluorescence confocal microscopy. Nile Red molecules were observed to associate with and dissociate from individual DMPC and SOPC vesicles adsorbed on a glass surface, generating on-and-off fluctuations in a fluorescence signal representing a very low noise two-state trajectory. Off-time statistics were used to investigate the mean radius of the vesicles and the size distribution functions. The means of the on-time distributions of Nile Red in DMPC and SOPC vesicles were significantly different. The association and dissociation reactions of single Nile Red molecules with a vesicle have been studied. Features of the bimolecular interaction between the probe Nile Red and the vesicle were evaluated from the uncorrelated mean on-time and vesicle radius distributions, and the linear Nile Red concentration dependence of the mean off-time. Nile Red is shown to be a useful probe of the structural fluctuations and heterogeneity of these membrane structures, and it is a useful model with which to directly study a diffusion-influenced reversible bimolecular reaction.

Dimyristoylphosphatidylcholine↗

PubMed2006

Controlled bimolecular collisions allow sub-diffraction limited microscopy of lipid vesicles.

The concentration and vesicle size-controlled collisions of single molecules with target biological assemblies allow sub-diffraction limited optical images to be obtained that are not subject to the usual photobleaching problems with single molecule experiments. For example, single molecules of the probe Nile Red in aqueous solution emit a burst of fluorescence when they collide with a 50 nm hydrophobic vesicle situated on the surface in the laser focus. The bimolecular kinetics of the bursts is defined by their on- and off-time distribution functions which depend on the concentration and diffusion of the probe and the vesicle size. The mean burst frequency changes much more sharply than does the fluorescence intensity when a vesicle is raster scanned through the laser focus. This sharpness allows the spatial resolution of two objects to be improved and separations less than the diffraction limited resolution of the conventional optical microscope to be measured. The principle of this method of trajectory time distribution optical microscopy (TTDOM) could be used in a far field optical microscopic system with a resolution of several nanometers.

Computer Simulation↗

PubMed2006

Preassembly and ligand-induced restructuring of the chains of the IFN-gamma receptor complex: the roles of Jak kinases, Stat1 and the receptor chains.

We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-gamma) receptor complex is preassembled (1). In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-gammaR1 results in a conformational change localized to IFN-gammaR1. Jak1 but not Jak2 is required for the two chains of the IFN-gamma receptor complex (IFN-gammaR1 and IFN-gammaR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-gammaR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-gammaR2 with IFN-gammaR1. These results agree with a detailed model of the IFN-gamma receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Amino Acid Sequence↗

PubMed2005

Interactions among the components of the interleukin-10 receptor complex.

We used fluorescence resonance energy transfer previously to show that the interferon-gamma (IFN-gamma) receptor complex is a preformed entity mediated by constitutive interactions between the IFN-gammaR2 and IFN-gammaR1 chains, and that this preassembled entity changes its structure after the treatment of cells with IFN-gamma. We applied this technique to determine the structure of the interleukin-10 (IL-10) receptor complex and whether it undergoes a similar conformational change after treatment of cells with IL-10. We report that, like the IFN-gamma receptor complex, the IL-10 receptor complex is preassembled: constitutive but weaker interactions occur between the IL-10R1 and IL-10R2 chains, and between two IL-10R2 chains. The IL-10 receptor complex undergoes no major conformational changes when cells are treated with cellular or Epstein-Barr viral IL-10. Receptor complex preassembly may be an inherent feature of Class 2 cytokine receptor complexes.

Animals↗

PubMed2003

Direct observation of triplet state emission of single molecules: single molecule phosphorescence quenching of metalloporphyrin and organometallic complexes by molecular oxygen and their quenching rate distributions.

Single molecules are detected through the phosphorescence emission of their triplet states. Emission of the triplet states of single molecules of Pt octabutoxycarbonyl porphyrin (PtOBCP) and ruthenium(II)-tris-4,7-diphenyl-1,10-phenanthroline dichloride (Ru(dpp)(3)Cl(2)) is reported. The single molecule phosphorescence is very sensitive to molecular oxygen. Each molecule has its own characteristic quenching rate by oxygen, and the distribution of these rates is measured for (Ru(dpp)(3)Cl(2)) on a quartz surface. The large variance of this distribution is presumed to be caused by fluctuations in the pseudobimolecular rate coefficient and the local oxygen concentration. The possibility of creating a quantitative single oxygen molecule sensor is suggested.

Luminescent Measurements↗

PubMed2003

Motions of single molecules and proteins in trehalose glass.

The fluorescence intensity-time records of individual metal-free porphyrin cytochrome-c and Zn porphyrin cytochrome-c molecules whose translational motions are restricted by encapsulation in trehalose are examined by single-molecule spectroscopy by means of a two-channel confocal microscope that records transient fluorescence signals in two orthogonal polarization directions. Large angular motions often occur on time scales ranging to many seconds. Measurements of the photobleaching time distributions indicate that the trehalose glass restricts the accessibility of the fluorescent molecules to oxygen.

Cytochrome c Group↗

PubMed2002

Seeing the light: preassembly and ligand-induced changes of the interferon gamma receptor complex in cells.

Our experiments were designed to test the hypothesis that the cell surface interferon gamma receptor chains are preassembled rather than associated by ligand and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer, a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation, and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand interferon gamma. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

Animals↗