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Results for “Single Molecule Imaging”

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At least 55 records · Page 3Linked to original sources
PubMed2000

Single-molecule imaging of interaction between dextran and glucosyltransferase from Streptococcus sobrinus.

Using total internal reflection fluorescence microscopy, we directly observed the interaction between dextran and glucosyltransferase I (GTF) of Streptococcus sobrinus. Tetramethylrhodamine (TMR)-labeled GTF molecules were individually imaged as they were associating with and then dissociating from the dextran fixed on the glass surface in the evanescent field. Similarly dynamic behavior of TMR-labeled dextran molecules was also observed on the GTF-fixed surface. The duration of the stay on the surface (dwell time) was measured for each of these molecules by counting the number of video frames that had recorded the image. A histogram of dwell time for a population of several hundred molecules indicated that the GTF-dextran interaction obeyed an apparent first-order kinetics. The rate constraints estimated for TMR-labeled GTF at pH 6.8 and 25 degrees C in the absence and presence of sucrose were 9.2 and 13.3 s(-1), respectively, indicating that sucrose accelerated the dissociation of GTF from dextran. However, the accelerated rate was still much lower than the catalytic center activity of GTF (> or = 25 s(-1)) under comparable conditions.

Dextrans↗
PubMed2003

Optical bioimaging: from living tissue to a single molecule: imaging and functional analysis of blood flow in organic microcirculation.

Activity of blood cells, erythrocytes, leucocytes, and platelets, in microcirculation was observed by using an intravital microscope and confocal laser scanning microscope connected with an image processing system including fluorescence and phosphorescence emission methods. Dynamic functions of the blood flow were mainly observed in mesentery, brain, and liver tissues of rats. The results are summarized as follows: Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, particularly at high shear rate. The spring constant and Young's modulus of diabetic erythrocytes obviously stiffened, making them hard to deform in the capillary. During hemorrhagic shock and thrombosis, flow velocity and oxygen partial pressure of blood decreased in the brain and liver tissues that can be visualized by using FITC stained erythrocytes and Pd-porphyrin derivative as a pO(2) probe. Platelet adhesion and thrombus formation in the micro-vessels accelerated under the photodynamic reaction; diabetic platelets showed augmented adhesion and aggregation on the vessel wall which was followed by acute thromboembolism. Active oxygen radicals take part in thrombus formation, accompanied with adhesion of the activated leucocytes. Fluorescent dye probes, rhodamine G and acridine orange, are quite useful for visualization of the flow behavior of platelets and leucocytes, respectively.

Animals↗
PubMed2006

Functionalized self-assembled monolayers on ultraflat gold as platforms for single molecule force spectroscopy and imaging.

Single molecule force spectroscopy is a valuable tool for studying unfolding and nanomechanical properties of proteins. The common practice is to stretch proteins from a surface that was dosed to give a reasonable hit rate and to analyze the curves that exhibit the expected characteristics of a single polymer. Whether the surface-bound proteins are indeed single and isolated remains unclear, and the undesirable protein/surface interactions that obscure informative features of the force curves are implicitly assumed to be absent. In this study, mixed self-assembled monolayers (SAMs) consisting of N-hydroxysuccinimide (NHS) and oligoethylene glycol (OEG) terminated thiols on an ultraflat gold surface were used to covalently immobilize proteins via lysine residues. By the optimization of attachment sites via lysine-NHS linkages amidst a protein-resistant layer of the OEG SAM, it was possible to isolate single proteins for study in a controlled fashion. The single protein distribution on the surface is clearly demonstrated by atomic force microscopy (AFM) imaging. The OEG also significantly reduces nonspecific tip-surface interactions between the cantilever and surface. Stretching covalently attached single proteins produces high-quality and reproducible force-extension curves. This experimental strategy is an attractive platform with which to study protein structure, interactions, and nanomechanical properties of single proteins.

Gold↗
PubMed1998

Scanning near-field optical microscopy in cell biology and microbiology.

Scanning a point light source in close proximity over a sample and recording the scattered or transmitted light intensity point by point allows one to record optical images with a resolution not limited by diffraction. An overview of this technique called scanning near-field optical microscopy (SNOM or NSOM) is given with emphasis on cell- and microbiology. After an introduction, where the basic features of the technique are explained, illustrative examples are presented, such as a HeLa cell, fluorescence labelled human chromosomes, super resolution fluorescence imaging, single molecule imaging and fluorescence resonance energy transfer between a single pair of dye molecules.

Chromosomes, Human↗
PubMedDate not supplied

Field enhancement and apertureless near-field optical spectroscopy of single molecules.

We report on fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. Our apertureless microscope is designed around a confocal fluorescence microscope associated with an AFM head. First, we show that the confocal microscope alone allows single molecule imaging and single molecule fluorescence analysis. When associated with the AFM head, we demonstrate, for the first time to our knowledge, that single molecule fluorescence is enhanced under the silicon tip. We tentatively attribute this effect to field enhancement under the tip.

Journal Article↗
PubMed1999

Single-molecule anisotropy imaging.

A novel method, single-molecule anisotropy imaging, has been employed to simultaneously study lateral and rotational diffusion of fluorescence-labeled lipids on supported phospholipid membranes. In a fluid membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, in which the rotational diffusion time is on the order of the excited-state lifetime of the fluorophore rhodamine, a rotational diffusion constant, D(rot) = 7 x 10(7) rad(2)/s, was determined. The lateral diffusion constant, measured by direct analysis of single-molecule trajectories, was D(lat) = 3.5 x 10(-8) cm(2)/s. As predicted from the free-volume model for diffusion, the results exhibit a significantly enhanced mobility on the nanosecond time scale. For membranes of DPPC lipids in the L(beta) gel phase, the slow rotational mobility permitted the direct observation of the rotation of individual molecules characterized by D(rot) = 1.2 rad(2)/s. The latter data were evaluated by a mean square angular displacement analysis. The technique developed here should prove itself profitable for imaging of conformational motions of individual proteins on the time scale of milliseconds to seconds.

Anisotropy↗
PubMed2002

Measuring the number of independent emitters in single-molecule fluorescence images and trajectories using coincident photons.

A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

Journal Article↗
PubMed1999

Fluorescence imaging of single molecules in polymer microspheres.

We report on far-field fluorescence imaging of single molecules in spherical polymer microparticles produced from solution by using microdroplet techniques. The fluorescence photobleaching quantum yields of rhodamine 6G in a common water-soluble polymer (polyvinyl alcohol) are at least five times smaller, corresponding to proportionally larger average fluorescence signals, than those in ethanolic solvents. This allows for acquisition of multiple images from a single molecule on a time scale of several minutes. We also show that fluorescent images of single molecules in microspheres can be calculated from semiclassic electrodynamics, which may ultimately be useful in retrieving dynamical information from experimental images.

Fluorescence↗
PubMed2001

Near-field fluorescence imaging of single molecules with a resolution in the range of 10 nm.

We demonstrate fluorescence imaging of single molecules, by near-field scanning optical microscopy (NSOM), using the illumination-collection mode of operation, with an aperture probe. Fluorescence images of single dye molecules were obtained with a spatial resolution of 15 nm, which is smaller than the diameter of the aperture (20 nm) of the probe employed. Such super-resolution may be attributable to non-radiative energy transfer from the molecules to the coated metal of the probe since the resolution obtained in the case of conventional NSOM is limited to 30-50 nm due to penetration of light into the metal.

Journal Article↗
PubMed2004

Single molecule fluorescence imaging and its application to the study of DNA condensation.

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.

DNA↗
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↗
PubMed2004

Single-molecule recognition imaging microscopy.

Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody-antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.

Animals↗
PubMed1996

Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.

Fluorescent Dyes↗
PubMed2005

Single-molecule fluorescence imaging of the remote TiO2 photocatalytic oxidation.

The remote TiO2 photocatalytic oxidation reaction of single dyes has been investigated by the single-molecule fluorescent imaging technique. The present results suggest that the active oxygen species (Ox) is most probably the .OH radical, which is generated from the photodecomposition of H2O2 by UV light. The analyses of the number, intensity, and spectrum of individual fluorescence spots at the single-molecule level also indicate that unoxidized and oxidized dyes exist during the bleaching processes of single dyes.

Journal Article↗