Michael George

Mapping the mechanical pulse of single cardiomyocytes with the atomic force microscope

Abstract The atomic force microscope (AFM) was used to analyse the contractile behaviour of embryonic chicken cardiomyocytes. The mechanical pulsing of cardiomyocytes was analysed by observing active single cells as well as cells in a confluent layer. When embedded in a confluent layer, owing to synchronisation, pulsing of the cells was often found to be very stable in terms of frequency and amplitude of the beat, including negative as well as positive amplitudes. Nevertheless, owing to movements of contraction centres within the layer, a flipping of the sign of the amplitude did sometimes also occur on a time scale of minutes. In contrast, single cells often changed between active periods of pulsing and periods of complete quietness. Also characteristic parameters like beat period and pulse amplitude were observed to be unstable. Finally, we combined the abilities of the AFM to image adherent single cells and to record locally beat amplitudes, to characterise the pulsing behaviour of single cells laterally resolved.

Activity of single ion channel proteins detected with a planar microstructure

We present recordings of currents mediated by single ion channel proteins obtained using planar, microstructured glass chips. In these chips, pores with diameters of 1–2 μm are produced by ion track etching and are used for patch clamping instead of using the classical micropipette. Our results represent success in using such devices to record from single channels in cell membranes. The planar chip greatly enhances the accessibility of the ion channel containing membrane and can serve as a workbench for experiments on single ion channels using combinations of patch clamp current recording with other single molecule techniques.

Investigation of the spatial resolution of the light-addressable potentiometric sensor

The spatial resolution of the light-addressable potentiometric sensor (LAPS) is investigated both theoretically and experimentally. For a theoretical analysis, the diffusion equation for minority charge carriers in the semiconductor was solved. The results suggest that by thinning the semiconductor wafer, the spatial resolution of the LAPS is no longer limited by the bulk minority charge carrier diffusion length. Spatial resolution in the micrometer range should thus be possible. For an experimental analysis, the effective diffusion length of light-generated charge carriers parallel to the sensor surface was measured. The results show that by increasing the doping density and by thinning the semiconductor substrate, spatial resolution of about 15 μm is obtained.

A novel design of multi-light LAPS based on digital compensation of frequency domain

Abstract In this report, a method which can significantly increase the measurement rate of the light-addressable potentiometric sensor (LAPS) is suggested. By illuminating the LAPS simultaneously at several positions, of which each is illuminated with a light pointer modulated with a different frequency, the surface potential at all illuminated regions can be measured simultaneously by analyzing the resulting photocurrent. By using this method, the rate to obtain a complete image of the surface potential distribution across a LAPS wafer can be drastically increased compared to the conventional mode, in which images are obtained by scanning the surface with one single light pointer. The technical feasibility of such a device is discussed and test measurements are presented.

Osteoclast-independent bone resorption by fibroblast-like cells.

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.

Spatially resolved monitoring of cellular metabolic activity with a semiconductor-based biosensor

Metabolic activity of cultured cells can be monitored by measuring changes in the pH of the surrounding medium caused by metabolic products such as protons, carbon dioxide or lactic acid. Although many systems designed for this purpose have been reported, almost all of them are based on bulk measurements, where the average metabolic activity of all cells in contact with the device is recorded. Here, we report on a novel biosensor, based on a modified light-addressable potentiometric sensor (LAPS) device, which enables the metabolic activity of cultured cells to be measured with spatial resolution. This is demonstrated here by detecting the differential sensitivity to a cholinergic receptor agonist of two different co-cultured cellular populations. By making simultaneous measurements of the metabolic activity of different cell types seeded on different segments of one sensor, this device not only provides a rapid means of assessing cellular specificity of pharmaceutical compounds but also has the potential of being used to non-invasively monitor humoral as well as synaptic communication between different cell populations in co-culture. The temporal and spatial resolution of the device were investigated and are discussed.

The field-effect-addressable potentiometric sensor/stimulator (FAPS)—a new concept for a surface potential sensor and stimulator with spatial resolution

Abstract We propose a new concept for a surface potential detector with high spatial resolution and large number of addressable points. The so-called field-effect-addressable potentiometric sensor/stimulator (FAPS) uses the ability to control the resistance of FET-channels in semiconductor films with electric potentials. Making use of a grid-structure of perpendicular FET-channels and gate electrodes located below the FET-channels, it is possible to tune a single intersection into maximum sensitivity to electrical potentials located above the FET-channel. By covering the surface above the FET-channels of a GaAs based FAPS with less than 100 nm PMMA, a bio-compatible interface could be created. This suggests the ability to record extracellular potentials and apply extracellular stimuli to electrically excitable cells adherent to the FAPS surface.

Electrically Excitable Normal Rat Kidney Fibroblasts: A New Model System for Cell-Semiconductor Hybrids

In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.

Effects of semiconductor substrate and glia-free culture on the development of voltage-dependent currents in rat striatal neurones

Abstract. An essential requirement for successful long-term coupling between neuronal assemblies and semiconductor devices is that the neurones must be able to fully develop their electrogenic repertoire when growing on semiconductor (silicon) substrates. While it has for some time been known that neurones may be cultured on silicon wafers insulated with SiO2 and Si3N4, an electrophysiological characterisation of their development under such conditions is lacking. The development of voltage-dependent membrane currents, especially of the rapid sodium inward current underlying the action potential, is of particular importance because the conductance change during the action potential determines the quality of cell-semiconductor coupling. We have cultured rat striatal neurones on either glass coverslips or silicon wafers insulated with SiO2 and Si3N4 using both serum-containing and serum-free media. We here report evidence that not only serum-free culture media but also growth on semiconductor surfaces may negatively affect the development of voltage-dependent currents in neurones. Furthermore, using surface-charge measurements with the atomic force microscope, we demonstrate a reduced negativity of the semiconductor surface compared to glass. The reduced surface charge may affect cellular development through an effect on the binding and/or orientation of extracellular matrix proteins, such as laminin. Our findings therefore suggest that semiconductor substrates are not entirely equivalent to glass in terms of their effects on neuronal cell growth and differentiation.

Characterization of the field-effect addressable potentiometric sensor /FAPS

The field-effect addressable potentiometric sensor FAPS is a surface potential sensor with the option of high spatial resolution. It is based on the ability to control the resistance of field-effect channels in thin semiconductor films with electrical potentials. Compared to . arrays of standard field-effect transistors FET , the number of required leads is drastically reduced by arranging the field-effect channels and underlying gate electrodes in a grid structure. In order to investigate the practicability of the physical concept, a test structure was built by using the epitaxial lift-off technique. It was demonstrated that the concept of the FAPS can be put into practice. The frequency dependence of the test structure was analyzed experimentally and theoretically. At a time resolution of 3 kHz, the potential sensitivity was found to be better than 150 mV. q 2000 Elsevier Science S.A. All rights reserved.