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At least 145 records · Page 8Linked to original sources
PubMed2006

Controlling binding site densities on glass surfaces.

The density of surface-immobilized ligands or binding sites is an important issue for the development of sensors, array- or chip-based assays, and single-molecule detection methods. The goal of this research is to control the binding site density of reactive ligands on surfaces by diluting surface amine groups in self-assembled and cross-linked monolayers on glass prepared from solutions containing very low concentrations of (3-aminopropyl)triethoxysilane (APTES) and much higher concentrations of (2-cyanoethyl)triethoxysilane. The surface amine sites are suitable for attaching labels and ligands by reaction with succinimidyl ester reagents. Labeling the amine sites with fluorescent molecules and imaging the single molecules with fluorescence microscopy provides a means of determining the density of amine sites on the surface, which were incorporated into the self-assembled monolayer with micrometer spacings in proportion to the concentration of APTES in the synthesis. Biotin ligands were also bound to these surface amine sites using a succinimidyl ester linker, and the immobilized biotin was then reacted with either streptavidin-conjugated gold colloid particles or fluorescently labeled neutravidin. Imaging of these samples yields consistent amine and biotin site coverages, indicating that quantitative control and chemical conversion of binding sites can be achieved at very low (<10(-7)) fractions of a monolayer.

Amines↗
PubMed2004

Nanometer-localized multiple single-molecule fluorescence microscopy.

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.

DNA↗
PubMedDate not supplied

Imaging stretched single DNA molecules by pulsed-force-mode atomic force microscopy.

The effect of a surface water layer on DNA strands deposited on a substrate was studied by atomic force microscopy (AFM). DNA molecules were deposited and stretched on chemically modified glass coverslips by a molecular combing method. Lambda bacteriophage DNA molecules were aligned on the organosilane-modified substrate surfaces by chemical and physical adsorption during the molecular combing. The combed DNA molecules were observed in humidity-controlled air and in aqueous solutions by pulsed-force-mode AFM (PFM-AFM). Chemical modification of cantilevers with an Au-coated tip by organothiol compounds was also applied to DNA observation. Mapping adhesive forces in aqueous media was useful to discriminate chemically the DNA strands from the substrate surface. The results suggest that PFM-AFM can be used widely to image the stretched DNA molecules on the silane-modified substrates.

Adsorption↗
PubMed2006

Imaging of single mRNA molecules moving within a living cell nucleus.

In eukaryotic cells, pre-mRNAs are transcribed in the nucleus, processed by 5' capping, 3'-polyadenylation, and splicing, and exported to the cytoplasm for translation. To examine the nuclear mRNA transport mechanism, intron-deficient mRNAs of truncated beta-globin and EGFP were synthesized, fluorescently labeled in vitro, and injected into the nucleus of living Xenopus A6 cells. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2microm(2)/s, about 1/150 of that in water. The slow diffusion could not be explained by simple diffusion obeying the Stokes-Einstein equation, suggesting interactions of the mRNAs with nuclear components. The remaining mRNAs were stationary with an average residence time of about 30s, comparable to the time required for mRNA diffusion from the site of synthesis to nuclear pores.

Active Transport, Cell Nucleus↗
PubMed2001

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules.

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Animals↗
PubMed1999

Imaging of single DNA molecule: applications to high-resolution genomic studies.

Single molecule analysis of DNA has revealed new insights into its structural and physical properties. The application of new methods for manipulating and visualizing DNA has resulted in important advances in high-resolution physical mapping of the genome and quantitative cytogenetic studies of genomic abnormalities (Lichter 1997). Studies of single molecules of DNA have employed a variety of approaches including electron microscopy, atomic force microscopy, scanning-tunneling microscopy and fluorescence microscopy. A number of new technologies have recently been developed to exploit fluorescence microscopy's full potential for genomic analysis and the fine mapping of subtle genetic alterations. In the case of the latter application, particular emphasis has been placed on developing new methods for stretching DNA for high-resolution fluorescence in-situ hybridization studies. We have recently described a process called molecular combing according to which single DNA molecules bound by their extremities to a solid surface are uniformly stretched and aligned by a receding air/water interface (Bensimon et al. 1994). In the following, we will review recent developments concerning molecular combing and discuss its current and potential applications for the high-resolution mapping of the human genome, the detection and quantification of subtle genomic imbalances and the positional cloning of disease-related genes.

Chromosome Aberrations↗
PubMed2004

Imaging of single-molecule translocation through nuclear pore complexes.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

Antigens, Viral, Tumor↗
PubMed2006

A simple DNA stretching method for fluorescence imaging of single DNA molecules.

Stretching or aligning DNA molecules onto a surface by means of molecular combing techniques is one of the critical steps in single DNA molecule analysis. However, many of the current studies have focused on lambda-DNA, or other large DNA molecules. There are very few studies on stretching methodologies for DNA molecules generated via PCR (typically smaller than 20 kb). Here we describe a simple method of stretching DNA molecules up to 18 kb in size on a modified glass surface. The very low background fluorescence allows efficient detection of single fluorescent dye labels incorporated into the stretched DNA molecules.

DNA↗
PubMed1999

Dynamics of single dye molecules observed by confocal imaging and spectroscopy.

The fluorescence emission of single rhodamine dye molecules (rhodamine 6G and rhodamine 630) at room temperature was analyzed by using scanning confocal laser microscopy in conjunction with polarization analysis, fluorescence spectroscopy, time-resolved detection (minutes to microseconds), and excitation saturation. Results are presented and discussed 1) for samples with dye molecules at the glass-air interface and 2) covered with an additional thin protective polymer film (polyvinylbutyral). Under the polymer layer, the single-molecule fluorescence was more stable than the glass-air interface. This result may be explained by fewer spontaneous variations of the fluorescence rate, polarization changes, spectral shifts, and longer photochemical lifetimes.

Fluorescent Dyes↗
PubMed1989

Imaging of single uncoated DNA molecules by scanning tunneling microscopy.

Scanning tunneling microscope images of DNA molecules absorbed onto highly oriented pyrolytic graphite have been obtained. Three methods of deposition and sample preparation have been utilized. In the first method, a highly concentrated solution of DNA is sonicated, and a drop is deposited on freshly cleaved graphite. Under these conditions, the molecules tend to align in a parallel fashion, forming liquid-crystalline phases. In the second method, a solution of DNA is deposited directly on the graphite surface without sonication. In this case, ammonium acetate, a volatile salt, is used to decrease the amount of the residual salt crystals left after drying. In the third method, a solution containing lysed phage particles and DNA is adsorbed onto a graphite surface. The molecules are seen either isolated or in small bundles. The values of height, periodicity, and thickness observed and the handedness of the molecules are consistent with those expected for DNA. In all cases, the molecules were identified by their characteristic periodic structure and because, at higher magnification, no graphite-like structure was detectable on the surface of the molecules. Often the DNA molecules appear to adsorb in areas of the graphite that have many steps and defects. A mechanism that explains the magnitude of the tunneling currents measured in DNA is proposed. This mechanism, in turn, suggests a general method by which large insulating molecules can be rendered conductive.

DNA↗
PubMed2002

Optical far-field microscopy of single molecules with 3.4 nm lateral resolution.

Optical far-field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low-temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of approximately 10 microm(3), are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.

Journal Article↗
PubMed2003

Detecting and quantifying colocalization of cell surface molecules by single particle fluorescence imaging.

Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.

Animals↗
PubMed2004

Fluorescence imaging for monitoring the colocalization of two single molecules in living cells.

The interaction, binding, and colocalization of two or more molecules in living cells are essential aspects of many biological molecular processes, and single-molecule technologies for investigating these processes in live cells, if successfully developed, would become very powerful tools. Here, we developed simultaneous, dual-color, single fluorescent molecule colocalization imaging, to quantitatively detect the colocalization of two species of individual molecules. We first established a method for spatially correcting the two full images synchronously obtained in two different colors, and then for overlaying them with an accuracy of 13 nm. By further assessing the precision of the position determination, and the signal/noise and signal/background ratios, we found that two single molecules in dual color can be colocalized to within 64-100 nm (68-90% detectability) in the membrane of cells for GFP and Alexa633. The detectability of true colocalization at the molecular level and the erroneous inclusion of incidental approaches of two molecules as colocalization have to be compromised at different levels in each experiment, depending on its purpose. This technique was successfully demonstrated in living cells in culture, monitoring colocalization of single molecules of E-cadherin fused with GFP diffusing in the plasma membrane with single molecules of Alexa633 conjugated to anti-E-cadherin Fab externally added to the culture medium. This work established a benchmark for monitoring the colocalization of two single molecules, which can be applied to wide ranges of studies for molecular interactions, both at the levels of single molecules and collections of molecules.

Fluorescent Dyes↗
PubMed2004

Single-molecule high-resolution imaging with photobleaching.

Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a "gap" in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10-20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms.

Base Sequence↗
PubMed2005

Imaging and detecting molecular interactions of single transmembrane proteins.

Single-molecule atomic force microscopy (AFM) provides novel ways to characterize structure-function relationships of native membrane proteins. High-resolution AFM-topographs allow observing substructures of single membrane proteins at sub-nanometer resolution as well as their conformational changes, oligomeric state, molecular dynamics and assembly. Complementary to AFM imaging, single-molecule force spectroscopy experiments allow detecting molecular interactions established within and between membrane proteins. The sensitivity of this method makes it possible to detect the interactions that stabilize secondary structures such as transmembrane alpha-helices, polypeptide loops and segments within. Changes in temperature or protein-protein assembly do not change the position of stable structural segments, but influence their stability established by collective molecular interactions. Such changes alter the probability of proteins to choose a certain unfolding pathway. Recent examples have elucidated unfolding and refolding pathways of membrane proteins as well as their energy landscapes. We review current and future potential of these approaches to reveal insights into membrane protein structure, function, and unfolding as we recognize that they could help answering key questions in the molecular basis of certain neuro-pathological dysfunctions.

Animals↗
PubMed2026

High-Purity Monovalent Functionalization of Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) show promise for probing molecular interactions at single-molecule resolution, yet generating SWCNT populations bearing a single defined functional tag remains challenging because surface functionalization is inherently stochastic. Here, we present a batch-scale strategy to produce predominantly singly tagged SWCNTs by leveraging the stochastic adsorption of single-stranded DNA (ssDNA). Specifically, SWCNTs are dispersed using a mixture of unmodified ssDNA (um-ssDNA) and a minor fraction of modified ssDNA (m-ssDNA) carrying an affinity handle. We developed a probabilistic ssDNA-SWCNT binding model that predicts the distribution of m-ssDNA per nanotube as a function of the input minor-strand fraction p = m-ssDNA/total ssDNA, enabling selection of conditions that maximize single-tag purity. Using magnetic-bead capture via a biotin affinity interaction and subsequent release, we isolate SWCNTs with 97.6% predicted single-tag purity at 2% recovery. Single-molecule fluorescence imaging further supports predominantly single-label occupancy under the model-selected conditions. Thus, this approach provides a general route to SWCNTs bearing a single molecular handle for downstream conjugation and assembly, supporting diverse future applications in SWCNT-based nanotechnologies.

Nanotubes, Carbon↗