Atomic force microscopy investigation of isolated virions of murine leukemia virus

Virions of mouse leukemia virus spread on glass substrates were visualized by atomic force microscopy. The size distribution mode was 145 nm, significantly larger than that for human immunodeficiency virus particles. The distribution of particle sizes is broad, indicating that no two particles are likely identical in content or surface features. Virions possess knoblike protrusions, which may represent vestiges of budding from cell membranes. Particles which split open allowed imaging of intact cores with diameters of 65 nm. They also permitted estimation of viral shell thickness (35 to 40 nm) and showed the presence of a distinct trough between the shell and the core surface.     

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high-throughput analysis of mumps virus and a mumps virus-specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11-mercaptoundecanoic acid (MUA). Poly-L-lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly-L-lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 x 10(5) to 14 x 10(5) pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose-dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high-throughput analysis of intact virus and serodiagnosis of infectious diseases.     

Visualization of the structures of the hepatitis C virus replication complex

Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.     

Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 x 10(8) Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation.     

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.     

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.     

ATP‐dependent looping of DNA by ISWI

Snf2 related chromatin remodelling enzymes possess an ATPase subunit similar to that of the SF‐II helicases which hydrolyzes ATP to track along DNA. Translocation and any resulting torque in the DNA could drive chromatin remodeling. To determine whether the ISWI protein can translocate and generate torque, tethered particle motion experiments and atomic force microscopy have been performed using recombinant ISWI expressed in E. coli. In the absence of ATP, ISWI bound to and wrapped DNA thereby shortening the overall contour length measured in atomic force micrographs. Although naked DNA only weakly stimulates ATP hydrolysis by ISWI, both atomic force microscopy and tethered particle motion data indicate that the protein generated loops in the presence of ATP. The duration of the looped state of the DNA measured using tethered particle motion was ATP‐dependent. Finally, ISWI relaxed positively supercoiled plasmids visualized by atomic force microscopy. While other chromatin remodeling ATPases catalyze either DNA wrapping or looping, both are catalyzed by ISWI. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Atomic force microscopy-based single virus particle spectroscopy

Atomic force microscopy-based single virus particle force spectroscopy was developed using dielectrophoresis for fixing virions at the tip of an atomic force microscope (AFM) probe. Electron microscopic visualization was found to be necessary to prove the deposition of virus particles on the tip of the AFM probe, while fixation of single virions by incubating the tip with a virus suspension proved impossible. Force spectroscopy measurements were performed for the vaccinia virus, influenza virus, and bacteriophage AP22. ForceReader special software was designed for analyzing the force–distance curves.     

A new method for quantitative estimation of the virus particles number

The virus particles of live mumps virus vaccine widely used for vaccination in Russia have been detected and visualized by the atomic force microscopy. For quantitative estimation of the number of observed virus particles the special method has been developed. The presence of the vaccine virus protein component was tested by ELISA and dot-blot analysis. Using a quantitative real-time PCR assay the number of copies of viral RNA was estimated. The results of the quantitative estimation obtained by real-time PCR corresponded to the atomic force microscopy data.     

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.     

Localization and force analysis at the single virus particle level using atomic force microscopy

Atomic force microscopy (AFM) is a vital instrument in nanobiotechnology. In this study, we developed a method that enables AFM to simultaneously measure specific unbinding force and map the viral glycoprotein at the single virus particle level. The average diameter of virus particles from AFM images and the specificity between the viral surface antigen and antibody probe were integrated to design a three-stage method that sets the measuring area to a single virus particle before obtaining the force measurements, where the influenza virus was used as the object of measurements. Based on the purposed method and performed analysis, several findings can be derived from the results. The mean unbinding force of a single virus particle can be quantified, and no significant difference exists in this value among virus particles. Furthermore, the repeatability of the proposed method is demonstrated. The force mapping images reveal that the distributions of surface viral antigens recognized by antibody probe were dispersed on the whole surface of individual virus particles under the proposed method and experimental criteria; meanwhile, the binding probabilities are similar among particles. This approach can be easily applied to most AFM systems without specific components or configurations. These results help understand the force-based analysis at the single virus particle level, and therefore, can reinforce the capability of AFM to investigate a specific type of viral surface protein and its distributions.     

Resolving the nanoscale adhesion of individual gecko spatulae by atomic force microscopy

Animals that cling to walls and walk on ceilings owe this ability to micrometre and nanoscale attachment elements. The highest adhesion forces are encountered in geckoes, which have developed intricate hierarchical structures consisting of toes (millimetre dimensions), lamella (400-600microm size), setae (micrometre dimensions) and spatulae ( approximately 200nm size). Adhesion forces of setae on different substrates have previously been measured by a micro-electromechanical system technique. Here we report the first successful experiments in which the force-displacement curves were determined for individual spatulae by atomic force microscopy. The adhesion force for these smallest elements of the gecko's attachment system is reproducibly found to be about 10nN. This method sheds new light on the nanomechanisms of attachment and will help in the rational design of artificial attachment systems.     

Diffusion and chemical reaction rates with nonuniform enzyme distribution: an experimental approach

The study of the effects of nonuniform distributions of immobilized beta-galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two beta-galactosidase substrates: lactose and ONPG in a commercial silica-alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized beta-galactosidase of different diameter (1 x 10(-4) to 5 x 10(-3) m) as catalyst, employing a temperature of 40 degrees C for lactose and 25 and 40 degrees C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 x 10(-3) m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

Imaging nanometer-sized α-synuclein aggregates by superresolution fluorescence localization microscopy

The morphological features of α-synuclein (AS) amyloid aggregation in vitro and in cells were elucidated at the nanoscale by far-field subdiffraction fluorescence localization microscopy. Labeling AS with rhodamine spiroamide probes allowed us to image AS fibrillar structures by fluorescence stochastic nanoscopy with an enhanced resolution at least 10-fold higher than that achieved with conventional, diffraction-limited techniques. The implementation of dual-color detection, combined with atomic force microscopy, revealed the propagation of individual fibrils in vitro. In cells, labeled protein appeared as amyloid aggregates of spheroidal morphology and subdiffraction sizes compatible with in vitro supramolecular intermediates perceived independently by atomic force microscopy and cryo-electron tomography. We estimated the number of monomeric protein units present in these minute structures. This approach is ideally suited for the investigation of the molecular mechanisms of amyloid formation both in vitro and in the cellular milieu.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Bacterial protein patterning by micro-contact printing of PLL-g-PEG

Biomimetic micro-patterned surfaces of three S-layer (fusion) proteins, wild type (SbpA), enhanced green fluorescence protein (SbpA-EGFP) and streptavidin (SbpA-STV), were built by microcontact printing of poly-L-lysine grafted polyethylene glycol (PLL-g-PEG). The functionality of the adsorbed proteins was studied with atomic force microscopy and fluorescence microscopy. Atomic force microscopy (AFM) measurements showed that wild-type SbpA recrystallized on PLL-g-PEG free areas, while fluorescent properties of SbpA-EGFP and the interaction of SbpA-streptavidin heterotetramers with biotin were not affected due to the adsorption on the micro patterned substrates.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

A method for micrometer resolution patterning of primary culture neurons for SPM analysis

In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-day-old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.     

Force spectroscopy between acetylcholinesterase molecule and its natural substrate to study the effects of inhibitors and reactivators on enzyme activity

The force spectrum (FS) between acetylcholinesterase (AChE) molecule and its natural substrates acetylcholine (ACh) and the influences of AChE inhibitors and reactivators have been investigated with atomic force microscopy (AFM) at single molecule level in real-time. AChE and ACh were covalently immobilized onto the surfaces of gold-plated mica and Si3N4 tip of the atomic force microscope respectively. First, AChE was imaged in image mode of AFM and one of AChE molecules was selected as the center of the scanning. Then scanning mode was changed into force scanning mode and FS was recorded in a frequency of 5 x s(-1). Solutions of drugs or toxicants can be injected from the fluid-in tube of the fluid cell at any desired time. The FS between ideally immobilized normal AChE, Inhibited AChE or aged AChE and ACh each had their own shape features. The influences of drugs or toxicants on these features could be observed in real-time on the screen of the computer. These results demonstrated that AFM force spectroscopy could be used as a new method to study the effects of drugs and toxicants on the activity of the enzyme in pharmacology and toxicology.