Getting to the heart of cardiac remodeling; how collagen subtypes may contribute to phenotype.

The objective of this study was to investigate the nature and biomechanical properties of collagen fibers within the human myocardium. Targeting cardiac interstitial abnormalities will likely become a major focus of future preventative strategies with regard to the management of cardiac dysfunction. Current knowledge regarding the component structures of myocardial collagen networks is limited, further delineation of which will require application of more innovative technologies. We applied a novel methodology involving combined confocal laser scanning and atomic force microscopy to investigate myocardial collagen within ex-vivo right atrial tissue from 10 patients undergoing elective coronary bypass surgery. Immuno-fluorescent co-staining revealed discrete collagen I and III fibers. During single fiber deformation, overall median values of stiffness recorded in collagen III were 37±16% lower than in collagen I [p<0.001]. On fiber retraction, collagen I exhibited greater degrees of elastic recoil [p<0.001; relative percentage increase in elastic recoil 7±3%] and less energy dissipation than collagen III [p<0.001; relative percentage increase in work recovered 7±2%]. In atrial biopsies taken from patients in permanent atrial fibrillation (n=5) versus sinus rhythm (n=5), stiffness of both collagen fiber subtypes was augmented (p<0.008). Myocardial fibrillar collagen fibers organize in a discrete manner and possess distinct biomechanical differences; specifically, collagen I fibers exhibit relatively higher stiffness, contrasting with higher susceptibility to plastic deformation and less energy efficiency on deformation with collagen III fibers. Augmented stiffness of both collagen fiber subtypes in tissue samples from patients with atrial fibrillation compared to those in sinus rhythm are consistent with recent published findings of increased collagen cross-linking in this setting.

Optimization of acoustic coupling for bottom actuated scattering based subsurface scanning probe microscopy.

The characterization of buried nanoscale structures nondestructively is an important challenge in a number of applications, such as defect detection and metrology in the semiconductor industry. A promising technique is Subsurface Scanning Probe Microscopy (SSPM), which combines ultrasound with Atomic Force Microscopy (AFM). Initially, SSPM was used to measure the viscoelastic contrast between a subsurface feature and its surrounding medium. However, by increasing the ultrasonic frequency to >1 GHz, it has been shown that SSPM can also measure acoustic impedance based contrasts. At these frequencies, it becomes difficult to reliably couple the sound into the sample such that the AFM is able to pick up the scattered sound field. The cause is the existence of strong acoustic resonances in the sample, the transducer, and the coupling layer-the liquid layer used to couple the sound energy from the transducer into the sample-in combination with the nonlinearity of the tip-sample interaction. Thus, it is essential to control and measure the thickness of the coupling layer with nanometer accuracy. Here, we present the design of a mechanical clamp to ensure a stable acoustic coupling. Moreover, an acoustic method is presented to measure the coupling layer thickness in real-time. Stable coupling layers with thicknesses of 700 ± 2 nm were achieved over periods of 2-4 h. Measurements of the downmixed AFM signals showed stable signal intensities for >1 h. The clamp and monitoring method introduced here makes scattering based SSPM practical, robust, and reliable and enables measurement periods of hours.

Single molecule binding dynamics measured with atomic force microscopy.

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3 nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods.

Stability enhancement of an atomic force microscope for long-term force measurement including cantilever modification for whole cell deformation.

Atomic force microscopy (AFM) is widely used in the study of both morphology and mechanical properties of living cells under physiologically relevant conditions. However, quantitative experiments on timescales of minutes to hours are generally limited by thermal drift in the instrument, particularly in the vertical (z) direction. In addition, we demonstrate the necessity to remove all air-liquid interfaces within the system for measurements in liquid environments, which may otherwise result in perturbations in the measured deflection. These effects severely limit the use of AFM as a practical tool for the study of long-term cell behavior, where precise knowledge of the tip-sample distance is a crucial requirement. Here we present a readily implementable, cost effective method of minimizing z-drift and liquid instabilities by utilizing active temperature control combined with a customized fluid cell system. Long-term whole cell mechanical measurements were performed using this stabilized AFM by attaching a large sphere to a cantilever in order to approximate a parallel plate system. An extensive examination of the effects of sphere attachment on AFM data is presented. Profiling of cantilever bending during substrate indentation revealed that the optical lever assumption of free ended cantilevering is inappropriate when sphere constraining occurs, which applies an additional torque to the cantilevers "free" end. Here we present the steps required to accurately determine force-indentation measurements for such a scenario. Combining these readily implementable modifications, we demonstrate the ability to investigate long-term whole cell mechanics by performing strain controlled cyclic deformation of single osteoblasts.

A compact multipurpose nanomanipulator for use inside a scanning electron microscope.

A compact, two-stage nanomanipulator was designed and built for use inside a scanning electron microscope. It consists of a fine stage employing piezostacks that provide a 15 microm range in three dimensions and a coarse stage based on commercially available stick-slip motors. Besides the fabrication of enhanced probes for scanning probe microscopy and the enhancement of electron field emitters, other novel manipulation processes were developed, such as locating, picking up, and positioning small nanostructures with an accuracy of approximately 10 nm. In combination with in situ I-V experiments, welding, and etching, this results in a multipurpose nanofactory, enabling a new range of experiments.