Markus Ludwig

Sensing specific molecular interactions with the atomic force microscope

One of the unique features of the atomic force microscope (AFM) is its capacity to measure interactions between tip and sample with high sensitivity and unparalleled spatial resolution. Since the development of methods for the functionalization of the tips, the versatility of the AFM has been expanded to experiments where specific molecular interactions are measured. For illustration, we present measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. Here, we compare different techniques that allow for the in situ measurement of the absolute value of the spring constant of cantilevers.

Atomic force microscope imaging contrast based on molecular recognition

The contrast in atomic force microscope images arises from forces between the tip and the sample. It was shown recently that specific molecular interaction forces may be measured with the atomic force microscope; consequently, we use such forces to map the distribution of binding partners on samples. Here we demonstrate this concept by imaging a streptavidin pattern with a biotinylated tip in a novel imaging mode called affinity imaging. In this mode topography, adhesion, and sample elasticity are extracted online from local force scans. We show that this technique allows the separation of these values and that the measured binding pattern is based on specific molecular interactions.

Topography feedback mechanism for the scanning electrochemical microscope based on hydrodynamic forces between tip and sample

The distance between microelectrode and sample is a crucial parameter in scanning electrochemical microscopy. Here we report on a technique where the hydrodynamic coupling between tip and sample is employed to measure and control this distance. We built an apparatus, where a microelectrode is vibrated laterally and the damping of the amplitude upon approach to the surface is measured optically. We show that amplitudes of up to 5 μm are easily obtained in an aqueous environment and that amplitudes below 1 nm can be detected with phase sensitive amplifiers. We measured the different vibration modes of the electrodes and identified the ones best suited for distance measurements. On hard surfaces we found the characteristic decay length of the coupling to be less than 1 μm and, within our measured range, independent of frequency and amplitude of the vibration. Measurements on an elastic surface of known spring constant revealed that the vibrating electrode exerts a repulsive normal force on the sample which m...

Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces.

At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.

Differences in F9 and 5.51 cell elasticity determined by cell poking and atomic force microscopy

We studied the elasticity of both a wild type (F9) mouse embryonic carcinoma and a vinculin‐deficient (5.51) cell line, which was produced by chemical mutagenesis. Using cell poking, we measured the effects of loss of vinculin on the elastic properties of these cells. F9 cells were about 20% more resistant to indentation by the cell poker (a glass stylus) than were 5.51 cells. Using the atomic force microscope to map the elasticity of wild type and vinculin‐deficient cells by 128×128 force scans, we observed a correlation of elasticity with cell poking elastometric measurements. These findings, as well as previous atomic force, rheologic, and magnetometric measurements [Goldmann and Ezzell, Exp. Cell Res. 226 (1996) 234–237; Ezzell et al., Exp. Cell Res. 231 (1997) 14–26], indicate that vinculin is an integral part of the cytoskeletal network.

Effect of the Sol-Gel Matrix on the Performance of Ammonia Fluorosensors Based on Energy Transfer

We report on the effect of organomodified sol-gel materials on fluorosensors for ammonia. The fluorosensors are based on ion pairs composed of an inert fluorophore and a pH-sensitive absorber dye and are embedded in sol-gel glass. Upon exposure to ammonia, deprotonation of the pH-sensitive dye bromophenolblue occurrs, and consequently, energy is transferred from the fluorophore rhodamine B or tetramethylrhodamine ethyl ester to the absorber. The response of the fluorosensors using different ratios of precursors, such as tetramethoxysilane and phenyltrimethoxysilane, is investigated. Detection limits for sol-gel layers composed of 50% tetramethoxysilane and 50% phenyltrimethoxysilane are as low as 0.1 mg/L of aqueous ammonia. Response times are of the order of 3 to 6 min for forward response. The reversibility of the sensor is related to the composition of the organically modified sol-gel glasses and is fastest for sensor layers composed of pure phenyltrimethoxysilane. Conditioning, regeneration, and storage of the layers are shown to be of vital importance for the performance of the sensor layers.

Probing the forces between complementary strands of DNA with the atomic force microscope

The atomic force microscope (AFM) is capable of measuring the interaction between tip and sample with high sensitivity and unparalleled spatial resolution. The chemical functionalization of the AFM tips has expanded the versatility of the AFM to experiments where specific molecular interactions are measured. We present here measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. We report a method that allows for the in situ measurement of the absolute value of the spring constant of cantilevers based on spectral analysis of the thermal excitations of the cantilever.