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Results for “Single Molecule Imaging”

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At least 163 records · Page 9Linked to original sources

PubMed2005

Real-time dipole orientational imaging as a probe of ligand-protein interactions.

Single-molecule orientational imaging using total internal reflection fluorescence microscopy has been employed to investigate the dynamics of a protein-ligand system. Emission patterns from single tetramethylrhodamine (TMR)-biocytin molecules bound to streptavidin show that the TMR dipole adopts a limited number of favored orientations. The angular trajectories of individual dipoles exhibit remarkably similar patterns that are characteristic of single TMR molecules interacting with a relatively homogeneous population of nanoenvironments. Analysis of the polar and azimuthal angle distributions reveals a tendency for the dipole to assume three primary and two secondary orientations. Autocorrelation analysis of the dipole trajectories shows a predominantly bimodal behavior in the reorientation rates with the slow and fast components corresponding to the primary and secondary orientations, respectively. A number of mechanisms by which the observed orientations might be stabilized have been considered, in particular specific interactions between the zwitterionic TMR probe and charged residues on the streptavidin surface. Variations in the reorientation rates have been discussed in terms of local thermal fluctuations in the protein.

Crystallography, X-Ray↗

PubMed2005

Single-molecule live-cell imaging of clathrin-based endocytosis.

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.

Adaptor Protein Complex 2↗

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↗

PubMed1984

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain.

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.

Animals↗

PubMed2003

Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy.

The detection of single mRNA molecules tagged by microinjected, singly fluorescently labeled oligo(dT) 43-mer molecules in living cells in quasi-natural surrounding, that is, cell culture medium, is demonstrated. Single-stranded oligonucleotides were labeled at the 5'-end with a red-absorbing oxazine derivative (MR121) and excited by a pulsed laser diode emitting at 635 nm with a repetition rate of 64 MHz. Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) on untreated living 3T3 mouse fibroblast cells reveals autofluorescence signals found predominately in the cytoplasm with fluorescence lifetimes of approximately 1.3 ns and emission maximums of approximately 665-670 nm. Hence, fluorescence signals of single MR121-labeled oligonucleotide molecules that exhibit a fluorescence lifetime of 2.8 ns and a fluorescence emission maximum of 685 nm can be easily discriminated against autofluorescence. MR121-labeled oligonucleotides were microinjected into the cytoplasm or nucleus of living 3T3 mouse fibroblast cells using a micropipet. Since the micropipet exhibits an inner diameter of 500 +/- 200 nm at the very end of the tip-comparable to the diameter of the detection volume applied-the number of molecules delivered into the cell via the micropipet can be counted. Furthermore, the presented technique enables the quantitative detection and time-resolved identification of single molecules in living cells as a result of their characteristic emission maximums and fluorescence lifetime. The results obtained from single-molecule studies demonstrate for the first time that 10-30% of the microinjected oligo(dT) 43-mer molecules cannot diffuse freely inside of the nucleus but, rather, are tethered to immobile elements of the transcriptional, splicing, or polyadenylation machinery.

Journal Article↗

PubMed2005

Motion of single DNA molecules at a liquid-solid interface as revealed by variable-angle evanescent-field microscopy.

A variable-angle total-internal-reflection fluorescence microscope (VATIRFM) capable of providing a large range of incident angles was constructed for imaging single DNA molecule dynamics at a solid/liquid interface. An algorithm using a public-domain image-processing program, ImageJ, was developed for single-molecule counting. The experimental counts at various incident angles with different evanescent-field layer (EFL) thicknesses are affected by molecular diffusion. The dynamics of molecules near the surface and the observed counts in the VATIRFM are elucidated using a limited one-dimensional random-walk diffusion model. The simulation fits well with the experimental counting results. Further analysis using the simulation reveals the details of single-molecule motion. One implication is that the measured intensities cannot be used directly to determine the distances of molecules from the surface, though the majority of fluorescence does come from the EFL. Another implication is that rather than providing molecular concentrations within EFL the experimental counting results depict the distance-dependent dynamics of molecules near the surface. Thus, the VATIRFM could be a powerful technique to study the surface repulsion/attraction of molecules within a few hundred nanometers of the surface. Further studies show that molecules at low ionic strengths experience electrostatic repulsion at distances much further away from the surface than the calculated thickness of the electrical double layer.

DNA↗

PubMed2005

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.

Biophysics↗

PubMed2000

Direct observation of single-molecule generation at a solid-liquid interface

Direct observation of single-molecule generation from a chemical reaction was achieved at a solid-liquid interface. The reaction between fluorescamine and immobilized N'-(3-trimethoxysilylpropyl)diethylenetriamine (DETA) was studied at the single-molecule level. Time-lapse fluorescence images of single-molecule products, excited by the evanescent field generated at a quartz-liquid interface, were recorded to follow the chemical reaction to its completion. The reactions were restricted to the approximately 1 nm thick layer nearest to the interface. Analysis of the photoelectron intensity of the fluorescent product of the reaction and its distribution shows that the reaction kinetics goes through a transition from zeroth-order to first-order as the reaction proceeds. This approach offered a novel means to study single-molecule reactions at the solid-liquid interface. It also enabled the investigation of reaction kinetics and chemical mapping of surface heterogeneity at the single-molecule level.

Journal Article↗

PubMed2006

Imaging and vibrational spectroscopy of single pyridine molecules on Ag(110) using a low-temperature scanning tunneling microscope.

A scanning tunneling microscope (STM) was used to extract the images of single, isolated pyridine molecules adsorbed on Ag(110) and to record their vibrational spectrum at 13 K. On the STM image, the pyridine molecule appears as an elongated protrusion along the [001] direction on top of a silver atom, indicating that it is bonded through its nitrogen lone pair electrons. STM inelastic electron tunneling spectroscopy of the adsorbed pyridine revealed C-D and C-H stretch modes at 282 and 378 meV, respectively.

Journal Article↗

PubMed2000

Imaging and tracking of single GFP molecules in solution.

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.

Biophysical Phenomena↗

PubMed2003

Near-field optical imaging of abasic sites on a single DNA molecule.

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.

Aldehydes↗

PubMed1999

Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.

The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions. Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays. In this study, we have imaged in real-time individual collagen I molecules and their proteolysis by Clostridium histolyticum collagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM). We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules. The proteolysis of collagen molecules was inhibited by reduced calcium and acidification. Results from AFM study of collagen proteolysis are consistent with SDS-PAGE biochemical assays. The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays. This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale. A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level. Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.

Aluminum Silicates↗

PubMed2005

Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules.

Tracking single molecules in the plasma membrane in live cells is becoming a useful technique for studying the spatial-temporal control of membrane molecular processes, such as signal transduction and the formation of large molecular complexes. In this review, three topics largely based on recent single-molecule observations are described, with a special emphasis on the results that are considered to be difficult to obtain using conventional methods monitoring the ensemble-averaged behavior of molecules. First, we describe the high-speed single-molecule tracking data, mostly obtained by our group that necessitated the paradigm shift of the plasma membrane structure, from the two-dimensional continuum fluid model to the compartmentalized fluid model. Second, we try to present a synthetic view of the cell membrane, which contains raft and other microdomains as well as being partitioned into small compartments. Furthermore, we present our working hypothesis, based on the literature, how large, stabilized rafts may be formed, after ligation or crosslinking, from small/unstable "reserve" rafts present in the steady-state cells. Finally, we explain our initial application of single-molecule fluorescence imaging for studies of the creation of T-cell receptor signaling complexes (immunological synapses or SMACS), by observing the recruitment of single Lck molecules as an initial approach. This revealed that the assembly of Lck at the T-cell receptor cluster site, observed by conventional fluorescence microscopy, actually represents dynamic concentrations of Lck molecules, entering and exiting the cluster domain rapidly, with the aid of the raft domains.

Animals↗

PubMed2005

Advances in fluorescence imaging with quantum dot bio-probes.

After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

Chromogenic Compounds↗

PubMed2004

Time-resolved confocal fluorescence imaging and spectrocopy system with single molecule sensitivity and sub-micrometer resolution.

We present novel technical features and results from a two channel confocal fluorescence lifetime microscope, which allows to efficiently investigate fluorescence dynamics down to the single molecule level. The MicroTime 200 time-resolved fluorescence microscope offers a multicolor excitation where different picosecond diode lasers are used. For imaging and positioning purposes we utilize a compact Piezo scanner which allows, due to a novel scanning algorithm and synchronisation technique, a superior movement and positioning accuracy. The data acquisition is completely based on time-correlated single photon counting, where every photon is detected and stored individually with its specific timing information (Time-Tagged Time-Resolved mode). This multiparameter data acquisition scheme offers the opportunity to analyse the parameter dependencies in a multitude of different ways. Standard intensity analysis can be used to reconstruct 2D-images or the temporal evolution (time trace) of the fluorescence of a single spot. The information from the two distinct detector channels additionally allows to investigate the polarisation of the emitted light or its spectral composition, for example for analysis of Fluorescence Resonance Energy Transfer (FRET). The timing information down to a picosecond scale offers the possibility not only to reconstruct fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function of any single spot of interest. The flexible multichannel detector scheme enables in this case also a cross-correlation between spectrally separated parts of the emission light, or even identical parts of the fluorescence to eliminate detector artifacts. The photon arrival coincidence analysis can also be expanded in the sub-ns range to study fluorescence antibunching in the fluorescence emission of single molecules. The ability of combining these different pieces of temporal information allows the construction of extremely powerful analysis methods and assays. We demonstrate a variety of these capabilities with results obtained from fluorescently labeled latex beads, biological samples, and single molecules excited in the blue or red wavelength region.

Equipment Design↗

PubMed2006

Enhanced fluorescence of Cy5-labeled DNA tethered to silver island films: fluorescence images and time-resolved studies using single-molecule spectroscopy.

Methods that increase the total emission per fluorophore would provide increased sensitivity and a wider dynamic range for chemical analysis, medical diagnostics, and in vivo molecular imaging. The use of fluorophore-metal interactions has the potential to dramatically increase the detectability of single fluorophores for bioanalytical monitoring. The fabrication and single-molecule analysis of fluorophore-labeled DNA molecules tethered to silver island films are described in this article. The single-molecule spectroscopic method reveals some insightful information on the behaviors of single molecules, rather than an ensemble of molecules. Analysis of fluorescence images, intensity profiles, total emitted photons, and lifetime distributions reveals some of sample heterogeneities. Investigations of time-dependent emission characteristics of single molecules indicate that the total number of emitted photons on the silvered surface is more than 10 times greater than on free labeled DNA molecules on a glass substrate. In addition, time-correlated single-photon counting results reveal the reduced lifetimes of single molecules tethered to silver island films.

Carbocyanines↗

PubMed1999

Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites.

Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of membrane electrophysiology in cells and cellular networks.

Animals↗

PubMed1990

Electron microscopy and image analysis of two-dimensional crystals and single molecules of alcohol oxidase from Hansenula polymorpha.

The octameric protein alcohol oxidase from the yeast Hansenula polymorpha was studied by electron microscopy and image analysis. Two-dimensional crystals were formed by applying the protein, in a phosphate buffer containing poly(ethylene glycol) and EDTA, to a carbon-coated formvar film which had been glow-discharged in pentylamine at least several hours earlier. The crystals show p4 symmetry and have a unit cell of 12.5 X 12.5 nm2, containing one molecule. Image analysis of the crystals and of single molecules yielded two different views. From these it can be deduced that the subunits have an elongated shape and form two layers of four, stacked face to face. A tentative model of the structure is presented.

Alcohol Oxidoreductases↗