Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations.

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca(2+)]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca(2+)]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K 1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

Minimum threshold for incipient plasticity in the atomic-scale nanoindentation of Au(111).

The formation of the smallest permanent indentation in a Au(111) surface is studied by scanning tunneling microscopy and atomic force microscopy in ultrahigh vacuum. The 9.5 nm radius W(111) indenter was characterized in situ by field ion microscopy. Elastic and plastic indentations are identified both in the residual impression image and by features in their force-displacement curves such as the sink-in depth, pop-ins, and hysteresis energy. Plasticity is best identified quantitatively in the force-displacement curves by the sink-in depth. The minimum of plastic damage producible in the substrate is associated with an energy budget of ∼70 eV.

Deconvolution of calcium fluorescent indicator signal from AFM cantilever reflection.

Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca²âº sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca²âº levels. When global Ca²âº responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.

The noise of coated cantilevers.

In atomic force microscopy, cantilevers with a reflective coating are often used to reduce optical shot noise for deflection detection. However, static AFM experiments can be limited by classical noise and therefore may not benefit from a reduction in shot noise. Furthermore, the cantilever coating has the detrimental side-effect of coupling light power fluctuations into true cantilever bending caused by time-varying thermal stresses. Here, we distinguish three classes of noise: detection, force, and displacement noise. We discuss these noises with respect to cantilever coating in the context of both static and dynamic AFM experiments. Finally, we present a patterned cantilever coating which reduces the impact of these noises.

Note: electrochemical etching of sharp iridium tips.

We describe an etching procedure for the production of sharp iridium tips with apex radii of 15-70 nm, as determined by scanning electron microscopy, field ion microscopy, and field emission measurements. A coarse electrochemical etch followed by zone electropolishing is performed in a relatively harmless calcium chloride solution with high success rate.

Switching atomic friction by electrochemical oxidation.

Friction between the sliding tip of an atomic force microscope and a gold surface changes dramatically upon electrochemical oxidation of the gold surface. Atomic-scale variations of the lateral force reveal details of the friction mechanisms. Stick-slip motion with atomic periodicity on perfect Au(111) terraces exhibits extremely low friction and almost no dependence on load. Significant friction is observed only above a load threshold at which wear of the surface is initiated. In contrast, irregular stick-slip motion and a linear increase of friction with load are observed on electrochemically oxidized surfaces. The observations are discussed with reference to the amorphous structure of the oxo-hydroxide surface and atomic place exchange mechanisms upon oxidation. Reversible, fast switching between the two states of friction has been achieved in both perchloric and sulfuric acid solutions.

Exploiting cantilever curvature for noise reduction in atomic force microscopy.

Optical beam deflection is a widely used method for detecting the deflection of atomic force microscope (AFM) cantilevers. This paper presents a first order derivation for the angular detection noise density which determines the lower limit for deflection sensing. Surprisingly, the cantilever radius of curvature, commonly not considered, plays a crucial role and can be exploited to decrease angular detection noise. We demonstrate a reduction in angular detection shot noise of more than an order of magnitude on a home-built AFM with a commercial 450 µm long cantilever by exploiting the optical properties of the cantilever curvature caused by the reflective gold coating. Lastly, we demonstrate how cantilever curvature can be responsible for up to 45% of the variability in the measured sensitivity of cantilevers on commercially available AFMs.

High-resolution friction force microscopy under electrochemical control.

We report the design and development of a friction force microscope for high-resolution studies in electrochemical environments. The design choices are motivated by the experimental requirements of atomic-scale friction measurements in liquids. The noise of the system is analyzed based on a methodology for the quantification of all the noise sources. The quantitative contribution of each noise source is analyzed in a series of lateral force measurements. Normal force detection is demonstrated in a study of the solvation potential in a confined liquid, octamethylcyclotetrasiloxane. The limitations of the timing resolution of the instrument are discussed in the context of an atomic stick-slip measurement. The instrument is capable of studying the atomic friction contrast between a bare Au(111) surface and a copper monolayer deposited at underpotential conditions in perchloric acid.