Categorization of two-photon microscopy images of human cartilage into states of osteoarthritis.

OBJECTIVE: The degeneration of articular cartilage is part of the clinical syndrome of osteoarthritis (OA) and one of the most common causes of pain and disability in middle-aged and older people(1). However, the objective detection of an initial state of OA is still challenging. In order to categorize cartilage into states of OA, an algorithm is presented which offers objective categorization on the basis of two-photon laser-scanning microscopy (TPLSM) images. METHODS: The algorithm is based on morphological characteristics of the images and results in a topographical visualization. This paper describes the algorithm and shows the result of a categorization of human cartilage samples. RESULTS: The resulting map of the analysis of TPLSM images can be divided into areas which correspond to the grades of the Outerbridge-Categorization. The algorithm is able to differentiate the samples in coincidence with the macroscopic impression. CONCLUSION: The method is promising for early OA detection and categorization. In order to achieve a higher benefit for the physician the method must be transferred to an endoscopic setup for an application in surgery.

Physisorbed surface coatings for poly(dimethylsiloxane) and quartz microfluidic devices.

Surface modifications of microfluidic devices are of essential importance for successful bioanalytical applications. Here, we investigate three different coatings for quartz and poly(dimethylsiloxane) (PDMS) surfaces. We employed a triblock copolymer with trade name F(108), poly(L-lysine)-g-poly(ethylene glycol) (PLL-PEG), as well as the hybrid coating n-dodecyl-ß-D-maltoside and methyl cellulose (DDM/MC). The impact of these coatings was characterized by measuring the electroosmotic flow (EOF), contact angle, and prevention of protein adsorption. Furthermore, we investigated the influence of static coatings, i.e., the incubation with the coating agent prior to measurements, and dynamic coatings, where the coating agent was present during the measurement. We found that all coatings on PDMS as well as quartz reduced EOF, increased reproducibility of EOF, reduced protein adsorption, and improved the wettability of the surfaces. Among the coating strategies tested, the dynamic coatings with DDM/MC and F(108) demonstrated maximal reduction of EOF and protein adsorption and simultaneously best long-term stability concerning EOF. For PLL-PEG, a reversal in the EOF direction was observed. Interestingly, the static surface coating strategy with F(108) proved to be as effective to prevent protein adsorption as dynamic coating with this block copolymer. These findings will allow optimized parameter choices for coating strategies on PDMS and quartz microfluidic devices in which control of EOF and reduced biofouling are indispensable.

Binding strength between cell adhesion proteoglycans measured by atomic force microscopy.

Measurement of binding forces intrinsic to adhesion molecules is necessary to assess their contribution to the maintenance of the anatomical integrity of multicellular organisms. Atomic force microscopy was used to measure the binding strength between cell adhesion proteoglycans from a marine sponge. Under physiological conditions, the adhesive force between two cell adhesion molecules was found to be up to 400 piconewtons. Thus a single pair of molecules could hold the weight of 1600 cells. High intermolecular binding forces are likely to form the basis for the integrity of the multicellular sponge organism.

Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies.

In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation of lambda- and T2-DNA (60 pM each) in periodically structured channels.

Absence of intrinsic electric conductivity in single dsDNA molecules.

The intrinsic dc conductivity of long, individual lambda phage dsDNA molecules has been investigated by ultrasensitive low current-voltage-spectroscopy (IV) under ambient conditions and controlled low humidity inert gas atmosphere on microfabricated metal-insulator-metal gap structures. We found a strong dependence of the measured conductivity on the apparent humidity, which we attribute to capillary condensation of water to the immobilized DNA molecules, giving rise to additional ionic currents. Additional IV-spectroscopy experiments under controlled argon atmosphere always revealed a significant drop in electrical conductivity to 4 x 10(-15)AV(-1)microm(-1), indicating almost no considerable contribution of electrical long range charge transport.

Large-scale homogeneous molecular templates for femtosecond time-resolved studies of the guest-host interaction.

Self-assembled monolayer films based on iodobenzoyloxy-functionalized resorc[4]arenes were prepared on gold substrates to serve as model systems for future time-resolved studies of molecular recognition, a mechanism of outstanding importance in bioorganic systems. The film properties were tested using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and imaging ellipsometry. An apparatus for time-resolved electron spectroscopy utilizing femtosecond soft X-ray pulses is capable of detecting iodine core-level photolines and the photoinduced dissociation after ultraviolet illumination. The developed technique holds promise for tracking the temporal evolution of chemical shifts of atomic markers as local probes for the dynamics of the guest-host interaction.

Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study.

The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein-DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoB(DBD)) was analyzed using surface plasmon resonance. In addition the two point mutants PhoB(DBD)D196A and PhoB(DBD)R219A were obtained and the DNA recognition in comparison to the wildtype PhoB(DBD) was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoB(DBD)-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoB(DBD). Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove.

Pulsed-field separation of particles in a microfluidic device.

We demonstrate the proof-of-principle of a new separation concept for micrometer-sized particles in a structured microfluidic device. Under the action of externally applied, periodic voltage-pulses two different species of like-charged polystyrene beads are observed to simultaneously migrate into opposite directions. Based on a theoretical model of the particle motion in the microdevice that shows good agreement with the experimental measurements, the underlying separation mechanism is identified and explained. Potential biophysical applications, such as cell sorting, are briefly addressed.

Analysis of subcellular surface structure, function and dynamics.

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).

Theoretical analysis of single-molecule force spectroscopy experiments: heterogeneity of chemical bonds.

We show that the standard theoretical framework in single-molecule force spectroscopy has to be extended to consistently describe the experimental findings. The basic amendment is to take into account heterogeneity of the chemical bonds via random variations of the force-dependent dissociation rates. This results in a very good agreement between theory and rupture data from several different experiments.

Probing single biomolecules with atomic force microscopy.

During the last years, atomic force microscopy (AFM) has developed from a microscopy tool for solid-state surface science toward a method employed in many scientific disciplines, such as biology, for investigating individual molecules on a nanometer scale. This article describes the current status of the imaging possibilities of AFM on RNA, IgG, and gold-labeled cell adhesion proteoglycans, as well as of measurements of intermolecular binding forces between biomolecules in order to investigate their molecular structure, function, and elasticity.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy.

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm-1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s-1 under zero force up to 15 s-1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Antigen binding forces of individually addressed single-chain Fv antibody molecules.

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.

A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.

Specific antigen/antibody interactions measured by force microscopy.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.

Supramolecular structure of a new family of circular proteoglycans mediating cell adhesion in sponges.

Aggregationfactors are the molecules responsible for species-specific cell adhesion in sponges. Here, we present the structure of the aggregation factor from the marine sponge Microciona prolifera, which constitutes the first description of a circular proteoglycan. We have analyzed chemically dissociated and enzymatically digested aggregation factor with atomic force microscopy, agarose gel electrophoresis, and Western blots using antibodies against the protein and carbohydrate moieties. Twenty units from each of two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms, respectively, stabilized by a hyaluronidase-sensitive component. MAFp3 carries a 200-kDa glycan involved in homologous self-interactions between aggregation factor molecules, whereas MAFp4 carries a 6-kDa glycan that binds cell surface receptors. A 68-kDa lectin found in cell membranes of several sponge species binds the aggregation factor and its protein-free glycans, as well as chondroitin sulfate and hyaluronan. Here, we show that despite their lack of clear sequence homologies with other known proteoglycan structures, the protein and carbohydrate components of sponge aggregation factors assemble to form a supramolecular complex remarkably similar to classical proteoglycans.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.