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Results for “Single Molecule Imaging”

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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↗

PubMed2004

Role of the membrane skeleton in creation of microdomains.

The membrane skeleton is a specialized part of the cytoskeleton that is in close proximity to the cell membrane with a protein composition and structure that differs from that of the bulk cytoskeleton. The membrane skeleton and various transmembrane proteins bound to it form a mosaic of compartments in the membrane that is responsible for the temporary confinement of membrane proteins and lipids and controls the rate of their repetitive hop movements between these membrane skeleton-based compartments, known as "hop diffusion", found by observation of single-molecule diffusion. Such hop diffusion has been found to be universal with compartment sizes that range from 30 to 700 nm, depending on the cell type. The part of the membrane skeleton that is directly involved in temporal confinement of membrane molecules has been successfully imaged by raster scanning a single membrane molecule using an optical trap (single molecule scanning optical force imaging). Such compartmentalization enables dynamic spatial regulation of signal transduction in the plasma membrane, by arresting signaling complexes of activated receptor molecules and enlarged, stabilized rafts within a compartment. Furthermore, high concentrations of the membrane skeleton and its associated immobile transmembrane proteins are involved in formation of the cell membrane polarity such as is found across the initial segment between the axon and the cell body in neurons. Argument is advanced that the creation of various membrane domains in the cell membrane must be influenced by the membrane skeleton.

Animals↗

PubMed2004

High-resolution scanning tunneling microscopy for molecules.

Scanning tunneling microscopy (STM) can detect individual molecular configuration with its high spatial resolution ability, but some intrinsical and extrinsic factors result in the complexities of STM imaging of single molecules. By combining STM experimental work and theoretical simulation with the local density approximation based on Bardeen perturbation method, we have explored the atomic-scale configuration of the following molecular systems: C(60) molecules adsorbed on Si(111)-(7x7); alkanethiol self-assembly monolayers on Au(111); C(60) molecule imaged by STM tip adsorbed with another C(60) molecule; O(2) molecule adsorbed on Ag(110) and CO molecule adsorbed on Cu(111) imaged by CO chemically modified STM tip. Some related problems including: molecule-substrate interactions, STM imaging mechanism, chemically modified STM tip, etc., are discussed.

Journal Article↗

PubMed2002

Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy.

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane alpha-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the alpha-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.

Bacteriorhodopsins↗

PubMed2000

Transmission Electron Diffraction at 200 eV and Damage Thresholds below the Carbon K Edge.

Transmission electron diffraction patterns from ultra-thin aromatic and aliphatic organic films at beam energies of 200 eV-1 keV have been recorded in a custom low energy electron transmission (LEET) chamber. A significant reduction of the molecular damage cross-section, measured by fading of diffraction spots, was found for thin films of the aromatic perylene when the beam energy was reduced from 400 to 200 eV. The corresponding measurements for the aliphatic tetracontane showed a smaller "threshold energy" and the differences are discussed. Electron beam damage from other aromatic materials has also been studied at low energy. Comparison of the carbon K shell ionization cross-section and the measured damage cross-sections show that carbon K-shell ionization is strongly correlated with the damage observed in aromatics at beam energies higher than 284 eV. Calculation of the minimum number of unit cells needed for imaging a single molecule, and comparison of calculated elastic with measured damage cross-sections both indicate new possibilities for imaging biomolecules with low energy electrons.

Journal Article↗

PubMed2004

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments.

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.

Calmodulin↗

PubMed2006

Single-molecule charge-transport measurements that reveal technique-dependent perturbations.

We compare scanning tunneling microscopy (STM) imaging with single-molecule conductive atomic force microscopy (C-AFM) measurements by probing a series of structurally related thiol-terminated oligo(phenylenevinylene)s (OPVs) designed to have unique charge-transport signatures. When one or two methylene spacers are inserted between the thiol points of attachment and the OPV core, a systematic reduction in the imaged molecular transconductance and the current transmitted through a metal-molecule-metal junction containing the molecule is observed, indicating good agreement between STM and C-AFM measurements. However, a structure where the OPV backbone is interrupted by a [2.2]paracyclophane core has a low molecular transconductance, as determined from STM images, and a high measured single-molecule conductance. This apparent disconnect can be understood by comparing the calculated molecular orbital topology of the OPV with one thiol bound to a gold surface (the geometry in the STM experiment) with the topology of the molecule with both thiol termini bound to gold (relevant to C-AFM). In the former case, a single contact splits low-lying molecular orbitals into two discrete fragments, and in the latter case, molecular orbitals that span the entire molecule are observed. Although the difference in observed conductance between the two different measurements is resolved, the overall set of observations highlights the importance of using combined techniques to better characterize charge-transport properties relevant to molecular electronics.

Journal Article↗

PubMed2001

Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy.

The spectral and photophysical characteristics of the autofluorescent proteins were analyzed and compared to flavinoids to test their applicability for single-molecule microscopy in live cells. We compare 1) the number of photons emitted by individual autofluorescent proteins in artificial and in vivo situations, 2) the saturation intensities of the various autofluorescent proteins, and 3) the maximal emitted photons from individual fluorophores in order to specify their use for repetitive imaging and dynamical analysis. It is found that under relevant conditions and for millisecond integration periods, the autofluorescent proteins have photon emission rates of approximately 3000 photons/ms (with the exception of DsRed), saturation intensities from 6 to 50 kW/cm2, and photobleaching yields from 10(-4) to 10(-5). Definition of a detection ratio led to the conclusion that the yellow-fluorescent protein mutant eYFP is superior compared to all the fluorescent proteins for single-molecule studies in vivo. This finding was subsequently used for demonstration of the applicability of eYFP in biophysical research. From tracking the lateral and rotational diffusion of eYFP in artificial material, and when bound to membranes of live cells, eYFP is found to dynamically track the entity to which it is anchored.

Bacterial Proteins↗

PubMed2006

Beyond Rayleigh's criterion: a resolution measure with application to single-molecule microscopy.

Rayleigh's criterion is extensively used in optical microscopy for determining the resolution of microscopes. This criterion imposes a resolution limit that has long been held as an impediment for studying nanoscale biological phenomenon through an optical microscope. However, it is well known that Rayleigh's criterion is based on intuitive notions. For example, Rayleigh's criterion is formulated in a deterministic setting that neglects the photon statistics of the acquired data. Hence it does not take into account the number of detected photons, which, in turn, raises concern over the use of Rayleigh's criterion in photon-counting techniques such as single-molecule microscopy. Here, we re-examine the resolution problem by adopting a stochastic framework and present a resolution measure that overcomes the limitations of Rayleigh's criterion. This resolution measure predicts that the resolution of optical microscopes is not limited and that it can be improved by increasing the number of detected photons. Experimental verification of the resolution measure is carried out by imaging single-molecule pairs with different distances of separation. The resolution measure provides a quantitative tool for designing and evaluating single-molecule experiments that probe biomolecular interactions.

Calibration↗

PubMed1984

Localization of lipoyl-bearing domains in the alpha-ketoglutarate dehydrogenase multienzyme complex.

The alpha-ketoglutarate dehydrogenase complex from Escherichia coli consists of a core component, dihydrolipoyl transsuccinylase (E2), to which are noncovalently bound 12 polypeptide chains each of alpha-ketoglutarate dehydrogenase and dihydrolipoyl dehydrogenase. E2 exists as a cube-shaped complex comprising 24 identical chains and may be resolved from the other two enzyme components. Limited digestion of E2 with trypsin quantitatively removes domains containing the lipoic acid cofactor while leaving the quaternary structure of the complex intact. Averages of native and trypsin-modified E2 were computed from images of single molecules obtained from electron micrographs of negatively stained specimens. The two averages were very similar and were in general agreement with a model determined previously by X-ray crystallography. However, detailed analysis of the difference image, obtained by subtracting the average of the trypsin-treated E2 from the native E2, showed extra stain-excluding regions along the edges of the native molecule which we interpret as representing the lipoyl-bearing domains. Micrographs of mixtures of native and modified E2 were also analyzed in order to rule out staining or electron-optical artifacts as accounting for the results. On the basis of these results along with other available structural information, we propose that one function of the lipoyl domains is to permit interactions between distantly separated lipoyl moieties in the E2 complex; this proposal also agrees with recent results of modeling studies of biochemical data [Hackert, M.L., Oliver, R.M., & Reed, L.J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2226-2230].

Acyltransferases↗

PubMed2000

High-resolution imaging of antibodies by tapping-mode atomic force microscopy: attractive and repulsive tip-sample interaction regimes.

A force microscope operated with an amplitude modulation feedback (usually known as tapping-mode atomic force microscope) has two tip-sample interaction regimes, attractive and repulsive. We have studied the performance of those regimes to imaging single antibody molecules. The attractive interaction regime allows determination of the basic morphologies of the antibodies on the support. More importantly, this regime is able to resolve the characteristic Y-shaped domain structure of antibodies and the hinge region between domains. Imaging in the repulsive interaction regime is associated with the irreversible deformation of the molecules. This causes a significant loss in resolution and contrast. Two major physical differences distinguish the repulsive interaction regime from the attractive interaction regime: the existence of tip-sample contact and the strength of the forces involved.

Aluminum Silicates↗

PubMed1998

Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy.

Proteins are usually identified by their molecular weights, and atomic force microscopy (AFM) produces images of single molecules in three dimensions. We have used AFM to measure the molecular volumes of a number of proteins and to determine any correlation with their known molecular weights. We used native proteins (the TATA-binding protein Tbp, a fusion protein of glutathione-S-transferase and the renal potassium channel protein ROMK1, the immunoglobulins IgG and IgM, and the vasodilator-stimulated phosphoprotein VASP) and also denatured proteins (the red blood cell proteins actin, Band 3 and spectrin separated by SDS-gel electrophoresis and isolated from nitrocellulose). Proteins studied had molecular weights between 38 and 900 kDa and were imaged attached to a mica substrate. We found that molecular weight increased with an increasing molecular volume (correlation coefficient = 0.994). Thus, the molecular volumes measured with AFM compare well with the calculated volumes of the individual proteins. The degree of resolution achieved (lateral 5 nm, vertical 0.2 nm) depended upon the firm attachment of the proteins to the mica. This was aided by coating the mica with suitable detergent and by imaging using the AFM tapping mode which minimizes any lateral force applied to the protein. We conclude that single (native and denatured) proteins can be imaged by AFM in three dimensions and identified by their specific molecular volumes. This new approach permits detection of the number of monomers of a homomultimeric protein and study of single proteins under physiological conditions at the molecular level.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2006

Engineering luminescent quantum dots for in vivo molecular and cellular imaging.

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

Animals↗

PubMed1999

Single-molecule behavior of monomeric and heteromeric kinesins.

Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.

Adenosine Triphosphatases↗

PubMed2002

High-resolution colocalization of single dye molecules by fluorescence lifetime imaging microscopy.

Conventional fluorescence microscopy can be used to determine the positions of objects in space when those objects are separated by distances greater than several hundred nanometers, as restricted by the diffraction limit of light. Fluorescence microscopy/spectroscopy based on fluorescence resonance energy-transfer techniques can be used to measure separation distances below approximately 10 nm. To fill the gap between these fundamental limits, we have developed an alternative technique for high-resolution colocalization of fluorescent dyes. The technique is based on fluorescence lifetime imaging. Under favorable conditions, the method can be used to distinguish, and to measure the distance between, two dye molecules that are less than 30 nm apart. To demonstrate the method, lifetime images of a mixture of Cy5 and JF9 (rhodamine derivative) molecules statistically adsorbed on a glass surface were acquired and analyzed. Since these two molecular species differ in fluorescence lifetime (for Cy5, tau(f) = 2.0 ns, and for JF9, tau(f) = 4.0 ns), it is possible to assign the contribution of fluorescence of the two dye types to each image pixel using a pattern recognition technique. Since both dye types can be excited using the same laser wavelength, the measurement is free of chromatic aberrations. The results presented demonstrate the first high-precision distance measurements between single conventional fluorescent dyes based solely on fluorescence lifetime.

Journal Article↗

PubMed1989

Symmetry and dimensions of membrane-bound nicotinic acetylcholine receptors from Torpedo californica electric tissue: rapid rearrangement to two-dimensional ordered lattices.

Computer-aided image-averaging methods are applied to different preparations of membrane-bound nicotinic acetylcholine receptor. Circular harmonic averaging (CHA), a novel, reference-independent averaging method developed by W. Kunath and H. Sack-Kongehl [1989) Ultramicroscopy 27:171-184) allows analyzing images of single molecules of the receptor in its native membrane-bound state. The five subunits of the receptor are clearly resolved. At the resolution obtained (approximately 20 A) no differences were observed with resting and agonist-desensitized receptors. A method is proposed for rapidly arranging the acetylcholine receptors to ordered lattices. Depending on the conditions, tetragonal or hexagonal, two-dimensional lattices can be obtained within 2 to 6 days at 4 degrees C. Analysis by CHA shows that the receptor molecules preserve their gross structure and dimensions in these membranes, but that they are randomly oriented. Both lattices, therefore, do not represent true two-dimensional crystals.

Animals↗

PubMed2002

Imaging of the fluorescence spectrum of a single fluorescent molecule by prism-based spectroscopy.

We have devised a novel method to visualize the fluorescence spectrum of a single fluorescent molecule using prism-based spectroscopy. Equipping a total internal reflection microscope with a newly designed wedge prism, we obtained a spectral image of a single rhodamine red molecule attached to an essential light chain of myosin. We also obtained a spectral image of single-pair fluorescence resonance energy transfer between rhodamine red and Cy5 in a double-labeled myosin motor domain. This method could become a useful tool to investigate the dynamic processes of biomolecules at the single-molecule level.

Carbocyanines↗

PubMed2001

Cryo-electron microscopy as an investigative tool: the ribosome as an example.

Cryo-electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three-dimensional reconstruction, cryo-EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA-protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X-ray structures of ribosomal subunits have become available, conformational changes observed by cryo-EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X-ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery.

Animals↗