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Results for “Single Molecule Imaging”

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At least 73 records · Page 4Linked to original sources

PubMed1996

Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels.

Individual fluorescent molecules and individual singly labeled proteins were observed in the water-filled pores of poly(acrylamide) gels by far-field microscopy. Brownian motion was markedly reduced by the gel framework, thus enabling extended study of single fluorophores in aqueous environments. A highly axially dependent laser field was used both to excite the fluorophores and to image the molecules in three dimensions. Single molecules were followed as they moved within and through the porous gel structure. In contrast to dry polymeric hosts, these water-based gels may form a useful medium for single-molecule studies of biological systems in vitro.

Acrylic Resins↗

PubMed2006

Nonblinking and long-lasting single-molecule fluorescence imaging.

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Carbocyanines↗

PubMed2005

Imaging of tautomerism in a single molecule.

Fluorescence imaging is used to visualize directly the transfer of two inner hydrogen atoms in single porphycene molecules. This reaction leads to a chemically equivalent but differently oriented structure and hence results in a rotation of the transition dipole moments. By probing single immobilized molecules with an azimuthally polarized laser beam in the focal spot of a confocal microscope we observe ring-like emission patterns, possible only for a chromophore with two nearly orthogonal transition dipole moments. Numerical simulations of the observed emission patterns yield a value of 72 degrees for the angle between the S0-S1 transition moments in the two tautomeric forms.

Journal Article↗

PubMed2005

Visualizing and manipulating individual protein molecules.

Single-molecule imaging and manipulation techniques have evolved in the past decade from mere jaw-dropping attractions to essential laboratory tools. By applying single-molecule methods important insights otherwise unavailable have been obtained on various biomolecular systems. Constantly improving single-molecule imaging techniques keep expanding the scale of the explorable spatial detail, thereby providing possible solutions to getting around the debilitating diffraction limit present in physiological-condition structural investigations. In some areas, such as motor protein studies, single-molecule methods have become part of the routine and essential research toolkit. Entire research fields, such as single-molecule force spectroscopy, have been born. In the present review single-molecule visualization and manipulation methods are reviewed with a focus on proteins. Relevant signals and prominent applications are discussed along with experimental examples and recent important results. Finally, the perspectives of the single-molecule field are explored.

Elasticity↗

PubMed2002

Stretching and imaging single DNA molecules and chromatin.

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion: understanding the mechanical properties of these molecules helps us understand how they function in the cell. The aim of this chapter is to summarize and critically evaluate the properties of single DNA molecules and of single chromatin fibers. The use of the high-resolution imaging capabilities of the atomic force microscopy has been covered, together with manipulating techniques such as optical fibers, optical and magnetic tweezers, and flow fields. We have learned a lot about DNA and how it responds to applied forces. It is also clear that even though the study of the properties of individual chromatin fibers has just begun, the single-molecule approaches are expected to provide a wealth of information concerning the mechanical properties of chromatin and the way its structure changes during processes like transcription and replication.

Animals↗

PubMed2006

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Animals↗

PubMed2000

Atomic force microscopy of native purple membrane.

Atomic force microscopy (AFM) allows the observation of surface structures of purple membrane (PM) in buffer solution with subnanometer resolution. This offers the possibility to classify the major conformations of the native bacteriorhodopsin (BR) surfaces and to map the variability of individual polypeptide loops connecting transmembrane alpha-helices of BR. The position, the variability and the flexibility of these loops depend on the packing arrangement of BR molecules in the lipid bilayer with significant differences observed between the trigonal and orthorhombic crystal forms. Cleavage of the Schiff base bond leads to a disassembly of the trigonal PM crystal, which is restored by regenerating the bleached PM. The combination of single molecule AFM imaging and single molecule force-spectroscopy provides an unique insight into the interactions between individual BR molecules and the PM, and between secondary structure elements within BR.

Bacteriorhodopsins↗

PubMed2006

Bright-field TEM imaging of single molecules: dream or near future?

We examine the suitability of spherical aberration (C(S))-corrected (CS) and uncorrected (UC) transmission electron microscopes (TEM) for conventional bright-field imaging of radiation-sensitive materials. We have chosen an individual molecule suspended in vacuum as a hypothetical example of a well-defined radiation-sensitive sample. We find that for this particular sample, CS instruments provide about 30% improvement over an UC instrument in terms of signal/noise ratio per unit electron dose at 300kV. The lowest imaging doses can be achieved in CS instruments equipped with high-brightness electron source operated at low incident electron energies. Our calculations suggest that it may be possible to image individual, iodine- or bromine-substituted organic molecules in bright-field mode, at doses lower than the accepted values for radiation damage of aromatic molecules.

Journal Article↗

PubMedDate not supplied

A structure for the signal sequence binding protein SRP54: 3D reconstruction from STEM images of single molecules.

The 54-kDa subunit SRP54 of the signal recognition particle in eukaryotic cells is responsible for the recognition of nascent proteins destined for secretion or membrane integration. The three-dimensional structure of this protein was determined using computational techniques applied to images of the molecule obtained via high-resolution, low-dose, scanning transmission electron microscopy at low temperature. The reconstructions at spatial resolutions between 12 and 15 A feature two unequal domains joined by a slender linker. The two-domain structure is in agreement with genetic and biochemical data indicating organization of SRP54 into a larger N-terminal GTP-binding region and a smaller C-terminal peptide-binding region. The structure has similarities to other protein domains with related functions and similar amino acid sequences. The larger domain of the 3D reconstruction is consistent in shape and size with the GTP-binding domains of EF-Tu and p21-RAS, while the smaller domain is compatible in structure with part of the peptide-binding protein calmodulin. The overall shape of SRP54 and the deduced location of critical functional regions of the molecule provide a structural framework for its known biochemical properties in the targeting cycle of the signal recognition particle.

Amino Acid Sequence↗

PubMed2001

An approach to three-dimensional structures of biomolecules by using single-molecule diffraction images.

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-A resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

Computer Simulation↗

PubMed1997

Single-molecule microscopy on model membranes reveals anomalous diffusion.

The lateral mobility of lipids in phospholipid membranes has attracted numerous experimental and theoretical studies, inspired by the model of Singer and Nicholson (1972. Science, 175:720-731) and the theoretical description by Saffman and Delbrück (1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113). Fluorescence recovery after photobleaching (FRAP) is used as the standard experimental technique for the study of lateral mobility, yielding an ensemble-averaged diffusion constant. Single-particle tracking (SPT) and the recently developed single-molecule imaging techniques now give access to data on individual displacements of molecules, which can be used for characterization of the mobility in a membrane. Here we present a new type of analysis for tracking data by making use of the probability distribution of square displacements. The potential of this new type of analysis is shown for single-molecule imaging, which was employed to follow the motion of individual fluorescence-labeled lipids in two systems: a fluid-supported phospholipid membrane and a solid polymerstabilized phospholipid monolayer. In the fluid membrane, a high-mobility component characterized by a diffusion constant of 4.4 microns2/s and a low-mobility component characterized by a diffusion constant of 0.07 micron2/s were identified. It is proposed that the latter characterizes the so-called immobile fraction often found in FRAP experiments. In the polymer-stabilized system, diffusion restricted to corrals of 140 nm was directly visualized. Both examples show the potentials of such detailed analysis in combination with single-molecule techniques: with minimal interference with the native structure, inhomogeneities of membrane mobility can be resolved with a spatial resolution of 100 nm, well below the diffraction limit.

Diffusion↗

PubMed1995

Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution.

Visualization of single actin filaments by fluorescence microscopy led to the development of new in vitro assays for analysing actomyosin-based motility at the molecular level. The ability to manipulate actin filaments with a microneedle or an optical trap combined with position-sensitive detectors has enabled direct measurements of nanometre displacements and piconewton forces exerted by individual myosin molecules. To elucidate how myosin generates movement, it is necessary to understand how ATP hydrolysis is coupled to mechanical work at the level of the single molecule. But the most sensitive microscopic ATPase assay available still requires over 1,000 myosins. To enhance the sensitivity of such assays, we have refined epifluorescence and total internal reflection microscopies to visualize single fluorescent dye molecules. We report here that this approach can be used directly to image single fluorescently labelled myosin molecules and detect individual ATP turnover reactions. In contrast to previously reported single fluorescent molecule imaging methods, which used specimens immobilized on an air-dried surface, our method allows video-rate imaging of single molecules in aqueous solution, and hence can be applied to the study of many types of enzymes and biomolecules.

Adenosine Triphosphate↗

PubMed2004

High-power blue/UV light-emitting diodes as excitation sources for sensitive detection.

With advances in III-V nitride manufacturing processes, high-power light-emitting diode (LED) chips in the blue and UV wavelengths are now commercially available at reasonable cost and can be used as excitation sources in optical sensing. We describe the use of these high-power blue and UV LEDs for sensitive fluorescence detection, including chip-based flow cytometry, capillary electrophoresis (CE), and single-molecule imaging. By using a blue LED with a focusable power of approximately 40 mW as the excitation source for fluorescent beads, we demonstrate a simple chip-based bead sorter capable of enriching the concentration of green fluorescent beads from 63% to 95%. In CE experiments, we show that a mixture of analyte solution containing 30 nM 6-carboxyrhodamine 6G and 10 nM fluorescein can be separated and detected with excellent signal-to-noise ratio (approximately 17 for 10 nM fluorescein) using the collimated emission from a blue LED; the estimated mass detection limit was approximately 200 zmol for fluorescein. We also demonstrated ultrasensitive fluorescence imaging of single rhodamine 123 molecules and individual lambda-DNA molecules. At a small fraction of the cost of an Ar+ laser, high-power blue and UV LEDs are effective alternatives for lasers and arc lamps in fluorescence applications that demand portability, low cost, and convenience.

Cells↗

PubMed2000

Orientational imaging of single molecules by annular illumination

The absorption dipole orientation of single fluorescent molecules is determined by mapping the spatial distribution of the squared electric field components in a high-numerical-aperture laser focus. Annular illumination geometry and the vicinity of a plane dielectric/air interface strongly enhance the longitudinal field component and the transverse fields perpendicular to the polarization direction. As a result, all three excitation field components in the focus are of comparable magnitude. The scheme holds promise to monitor rotational diffusion of single molecules in complex environments.

Journal Article↗

PubMed2003

Optical bioimaging: from living tissue to a single molecule: calcium imaging in blood vessel in situ employing two-photon excitation fluorescence microscopy.

Recent developments in optoelectronics permit real-time Ca(2+) imaging of thin planes within cells utilizing laser scanning confocal microscopy (LSCM). However, a major complication associated with this imaging system involves increased phototoxicity with improved spatiotemporal resolution. Two-photon excitation microscopy (TPEM) helps to minimize phototoxicity due to the restriction of this technique to the volume proximal to the geometric focus of the light. In this study, the capability of Ca(2+) imaging was investigated employing recently developed real-time TPEM, RTS2000MP (Bio-Rad, Tokyo) with a mode-locked Ti-sapphire laser. Z-axis resolution of RTS2000MP with high NA objectives defined as full-width at half maximum (FWHM) with a 0.5-microm fluorescent bead provided values nearly identical to those obtained with LSCM at a small pinhole (0.2 mm) (approximately 0.6 microm). When serial sectioning of 21 sequential images at 0.3-microm intervals in cultured endothelial cells loaded with calcein and tetramethyl-rhodamine methylester were performed with TPEM, the z-axis resolution was higher than that observed with LSCM; moreover, the photobleaching rate was significantly lower than that obtained with LSCM. Maximum fluorescence intensities were detected at 780 nm in excitation spectra of fluo-3 and fluo-4 Ca(2+)-sensitive probes with TPEM. Fluorescence images in mouse arterial endothelial cells loaded with fluo-4 could be clearly visualized by TPEM in situ. Application of acetylcholine caused oscillatory increase in [Ca(2+)](i) of endothelial cells; subsequently, relaxation along the major axis of smooth muscle cells was evident. Furthermore, consecutive long-lasting experiments could be repeated with identical response in the same microscopic field. In conclusion, fluorescence imaging employing TPEM is useful for Ca(2+) imaging in blood vessels in situ.

Animals↗

PubMed2005

Total internal reflection fluorescence microscopy in single molecule nanobioscience.

Recent development in total internal reflection fluorescence microscopy (TIRFM) has made it possible to directly monitor the behaviors of biomolecules at the single-molecule level both in vitro and in living cells. This technique has opened a new era in bioscience, so-called single-molecule nanobioscience. In this review, we will summarize the theoretical basis of TIRFM, explain the instruments of TIRFM for single-molecules imaging, and discuss some novel applications of TIRFM to single-molecule nanobioscience.

Equipment Design↗

PubMed2001

Imaging of single fluorescent molecules using video-rate confocal microscopy.

Single fluorescent molecules in aqueous solution were imaged for the first time at video-rate using Nipkow disk-type confocal microscopy. Performance of this method was evaluated by imaging single kinesin molecules labeled with fluorescent dyes of tetramethylrhodamine (TMR) or IC5. Photodecomposition lifetimes of the fluorophores were approximately 10 s for TMR and approximately 2 s for IC5 under the incident laser power of 0.5 W/mm(2). Both the fluorescence intensity and the photobleaching rate were proportional to the laser power from 0.65 to 3 W/mm(2). 2D sliding movement of single kinesin molecules along microtubules on glass surface and 3D Brownian motion of individual kinesin molecules in viscous solution could be observed using this microscopy. These results indicated that this method could be applicable to the study of single molecular events in living cells at real time.

Animals↗

PubMed2005

Probing strong adsorption of solute onto C18-silica gel by fluorescence correlation imaging and single-molecule spectroscopy under RPLC conditions.

Understanding molecular adsorption at a chromatographic interface is of great interest for addressing the tailing problem in chemical separations. Single-molecule spectroscopy and confocal fluorescence correlation imaging are used to study the adsorption sites of C(18) silica beads under RPLC chromatographic conditions. The experiments show that cationic molecule rhodamine 6G laterally diffuses through the chromatographic interface of a C(18) hydrocarbon monolayer and acetonitrile with occasional reversible strong adsorptions. Fluorescence correlation imaging extracts the rare strong adsorption events from large data sets, revealing that the strong adsorption sites are randomly distributed throughout the silica beads. Virtually every imaging pixel of silica beads adsorbs molecules. Single-molecule spectroscopy of the 584 strong adsorption events observed indicates that the strong adsorptions persist on the time scales from several milliseconds to seconds, having an average desorption time of 61 ms. The strong adsorption events are rare, comprising 0.3% of the total observation time. The sizes of strong adsorption sites are within the optical resolution of confocal imaging.

Acetonitriles↗