SEARCH · PubMed Health

Results for “Single Molecule Imaging”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMed2000

The 11 A resolution projection map of Na+/K+-ATPase calculated by application of single particle analysis to two-dimensional crystal images.

Two-dimensional (2D) crystals of Na+/K+-ATPase were vitrified and observed with an electron cryo-microscope that allowed specimen observation at liquid helium temperature. Images showing 19 A spots on the optical diffraction patterns were digitized and processed by a crystallographic method. After optimization of parameters for the image processing, the locally averaged images revealed that each 2D crystal contained heterogeneous molecules. Single particle analysis was therefore applied to the 2D crystal images to select homogeneous molecule images, and averaging the selected images significantly improved map quality. Five major peak densities were clearly resolved in the new projection map, while two were found previously.

Animals↗

PubMed2004

Simultaneous wide-field imaging and spectroscopy of localized fluorophores.

A method that combines fluorescence imaging and spectroscopy of single molecules at room temperature is presented. This approach allows us to identify a number of imaged molecules unequivocally by simultaneously recording their fluorescence emission spectra. Furthermore, their spectral characteristics not only allow us to separate different fluorescent labels quantitatively and qualitatively but also provide information on the microenvironment of the molecules. This new method was successfully tested on a system of yellow-green and red fluorescent 20-nm latex beads, and its usefulness in studies of biological systems was illustrated for a preparation of combined binary ratio labeling-fluorescence in situ hybridization-stained mouse chromosomes.

Animals↗

PubMed1983

Structural analysis of human and bovine alpha-fetoprotein by electron microscopy, image processing, and circular dichroism.

The images of human and bovine alpha-fetoprotein molecules have been enhanced by combining dark-field electron microscopy with a laser-assisted optical system. This system filters out random background noise while permitting true averaged signal reconstruction of the molecule. A single averaged molecular image was digitized into a matrix, each pixel being assigned a gray scale level to produce a relative mass map for each molecule. These maps were interpreted from the alpha-helix, beta-form, and random coil of the purified proteins as determined by circular dichroism. Results showed that both molecules are "U shaped", apparently monomeric, with outside dimensions of approximately 80 A. Both molecules have asymmetrical structural features, notably three mass dense regions at both extremities and at the vertex of the molecules. Circular dichroism data suggest a high degree of similar stabilized alpha-helix and extensive beta-form in these regions. Mass map analysis of hAFP correlates with the subdomains organized by disulfide bridges.

Animals↗

PubMed2003

Monitoring molecular beacon DNA probe hybridization at the single-molecule level.

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.

Biosensing Techniques↗

PubMed1999

Microscopy for recognition of individual biomolecules.

One frontier challenge in microscopy and analytical chemistry is the analysis of soft matter at the single molecule level with biological systems as most complex examples. Towards this goal we have developed two novel microscopy methods. Both employ highly specific molecular recognition schemes used by nature-the recognition of specific protein sites by antibodies and ligands. One method uses fluorescence labeled ligands for detecting single molecules in fluid systems like membranes (Fig. 1B). Unitary signals are reliably resolved even for millisecond illumination periods. The knowledge of the unitary signal from single molecules permits the determination of stoichiometries of component association (Fig. 3). Direct imaging of the diffusional path of single molecules became possible for the first time (Fig. 4). Using linear polarized excitation, the angular orientation of single molecules can be analyzed (single molecule linear dichroism, (Fig. 5), which opens a new perspective for detecting conformational changes of single biomolecules. In the other method, an antibody is flexibly linked to the tip of an atomic-force microscope. This permits the identification of receptors in multi-component systems. Molecular mapping of biosurfaces and the study of molecular dynamics in the ms to s range become possible with atomic force microscopy.

Antibodies↗

PubMed2002

Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells.

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.

Adenoviridae↗

PubMed2006

Counting single native biomolecules and intact viruses with color-coded nanoparticles.

Nanometer-sized particles such as semiconductor quantum dots and energy-transfer nanoparticles have novel optical properties such as tunable light emission, signal brightness, and multicolor excitation that are not available from traditional organic dyes and fluorescent proteins. Here we report the use of color-coded nanoparticles and dual-color fluorescence coincidence for real-time detection of single native biomolecules and viruses in a microfluidic channel. Using green and red nanoparticles to simultaneously recognize two binding sites on a single target, we demonstrate that individual molecules of genes, proteins, and intact viruses can be detected and identified in complex mixtures without target amplification or probe/target separation. Real-time coincidence analysis of single-photon events allows rapid detection of bound targets and efficient discrimination of excess unbound probes. Quantitative studies indicate that the counting results are remarkably precise when the total numbers of counted molecules are more than 10. The use of bioconjugated nanoparticle probes for single-molecule detection is expected to have important applications in ultrasensitive molecular diagnostics, bioterrorism agent detection, and real-time imaging and tracking of single-molecule processes inside living cells.

Base Sequence↗

PubMed2005

Visualization of single receptor molecules bound to human rhinovirus under physiological conditions.

Dynamic force microscopy (DFM) was used to image human rhinovirus HRV2 alone and complexed with single receptor molecules under near physiological conditions. Specific and site-directed immobilization of HRV2 on a model cell membrane resulted in a crystalline arrangement of virus particles with hexagonal symmetry and 35 nm spacing. High-resolution imaging of the virus capsid revealed about 20 resolvable structural features with 3 nm diameters; this finding is in agreement with protrusions seen by cryo-electron microscopy. Binding of receptor molecules to individual virus particles was observed after injection of soluble receptors into the liquid cell. Virus-receptor complexes with zero, one, two, or three attached receptor molecules were resolved. The number of receptor molecules associated to virions increased over time. Occasionally, dissociation of single receptor molecules from viral particles was also observed.

Capsid↗

PubMed1990

Imaging the membrane protein bacteriorhodopsin with the atomic force microscope.

The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.

Adsorption↗

PubMed2000

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy.

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.

Animals↗

PubMed2006

High-resolution STM imaging of novel poly(G)-poly(C) DNA molecules.

High-resolution scanning tunneling microscopy (STM) imaging of single double-stranded poly(G)-poly(C) DNA molecules, made by a novel synthesis method, shows the molecules morphology. The STM images reveal a periodic structure of approximately 4 nm, seen as repeating "bulbs" along the molecules. These "bulbs" are associated with the molecule helix (the major grooves). "Nicks", two per 100 nm on the average, are observed along the DNA as well. The STM measurements were supported by a morphological statistics of the DNA molecule groove length and apparent height relative to the surface.

DNA↗

PubMed2006

Particle image correlation spectroscopy (PICS): retrieving nanometer-scale correlations from high-density single-molecule position data.

A new data analysis tool that resolves correlations on the nanometer length and millisecond timescale is derived. This tool, adapted from methods of spatiotemporal image correlation spectroscopy, exploits the high positional accuracy of single-particle tracking. While conventional tracking methods break down if multiple particle trajectories intersect, our method works in principle for arbitrarily large molecule densities and diffusion coefficients as long as individual molecules can be identified. The method is computationally cheap and robust and requires no a priori knowledge about the dynamical coefficients, as opposed to other methods. We demonstrate the validity of the method by Monte Carlo simulations and by application to single-molecule tracking data of membrane-anchored proteins in live cells. The results faithfully reproduce those obtained by conventional tracking. Upon activation, a fraction of the small GTPase H-Ras is confined to domains of <200 nm diameter, which further substantiates the prediction that membrane organization is a determinant in cellular signaling.

Algorithms↗

PubMed2003

The power and prospects of fluorescence microscopies and spectroscopies.

Recent years have witnessed a renaissance of fluorescence microscopy techniques and applications, from live-animal multiphoton confocal microscopy to single-molecule fluorescence spectroscopy and imaging in living cells. These achievements have been made possible not so much because of improvements in microscope design, but rather because of development of new detectors, accessible continuous wave and pulsed laser sources, sophisticated multiparameter analysis on one hand, and the development of new probes and labeling chemistries on the other. This review tracks the lineage of ideas and the evolution of thinking that have led to the actual developments, and presents a comprehensive overview of the field, with emphasis put on our laboratory's interest in single-molecule microscopy and spectroscopy.

Equipment Design↗

PubMed1991

Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope.

The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2005

Secondary ion MS imaging of lipids in picoliter vials with a buckminsterfullerene ion source.

Investigation of the spatial distribution of lipids in cell membranes can lead to an improved understanding of the role of lipids in biological function and disease. Time-of-flight secondary ion mass spectrometry is capable of molecule-specific imaging of biological molecules across single cells and has demonstrated potential for examining the functional segregation of lipids in cell membranes. In this paper, standard SIMS spectra are analyzed for phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cholesterol, and sulfatide. Importantly, each of the lipids result in signature mass spectral peaks that allow them to be identified. These signature peaks are also useful for imaging experiments and are utilized here to simultaneously image lipids on a micrometer scale in picoliter vials. Because the low secondary ion signal achieved for lipids from an atomic primary ion source makes cell-imaging experiments challenging, improving signal with cluster primary ion sources is of interest. Here, we compare the secondary ion yield for seven lipids using atomic (Ga+ or In+) ion sources and a buckminsterfullerene (C60+) primary ion source. A 40-1000-fold improvement in signal is found with C60+ relative to the other two ion sources, indicating great promise for future cellular imaging applications using the C60+ probe.

Fullerenes↗

PubMed1999

Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Adsorption↗

PubMed2004

Noise bias in the refinement of structures derived from single particles.

One of the main goals in the determination of three-dimensional macromolecular structures from electron microscope images of individual molecules and complexes (single particles) is a sufficiently high spatial resolution, about 4 A, at which the interpretation with an atomic model becomes possible. To reach high resolution, an iterative refinement procedure using an expectation maximization algorithm is often used that leads to a more accurate alignment of the positional and orientational parameters for each particle. We show here the results of refinement algorithms that use a phase residual, a linear correlation coefficient, or a weighted correlation coefficient to align individual particles. The algorithms were applied to computer-generated data sets that contained projections from model structures, as well as noise. The algorithms show different degrees of over-fitting, especially at high resolution where the signal is weak. We demonstrate that the degree of over-fitting is reduced with a weighting scheme that depends on the signal-to-noise ratio in the data. The weighting also improves the accuracy of resolution measurement by the commonly used Fourier shell correlation. The performance of the refinement algorithms is compared to that using a maximum likelihood approach. The weighted correlation coefficient was implemented in the computer program FREALIGN.

Algorithms↗

PubMed1998

Three-dimensional image analysis of myosin head in function as captured by quick-freeze deep-etch replica electron microscopy.

Quick-freeze deep-etch replica electron microscopy combined with mica-flake technique provides high contrast, high time- and spatial-resolution images of protein molecules in solution, whose three-dimensional structure is well preserved. Thus, it might be quite useful to obtain structural information of individual functioning molecules, such as myosin crossbridges under in vitro motility assay conditions. With that method, we could actually show that both heads of heavy meromyosin (HMM) crossbridges are mostly straight and bound to actin filaments with about 45 degree tilt-angle under rigor conditions, whereas they attached to actin through only one head with a wide variety of angles under in vitro sliding conditions. We also demonstrated that free HMM heads are strongly kinked in the presence of ATP or ADP/inorganic vanadate (Vi) in contrast to almost straight configuration in the absence of nucleotide. To examine more detailed structure of individual crossbridges, we tried to reconstruct the three-dimensional architecture of intramolecular subdomains of single HMM molecule. We took a series of tilted images of single HMM-ADP/Vi particle and successfully obtained its 3-D image by filtered back-projection, even with restricted range of tilt-angles. By comparison of the reconstruction with the atomic model of subfragment-1 (S1) without nucleotide, we found some great structural difference, which partly might be attributable to the conformational change by nucleotide binding.

Actins↗