A scanning force microscope for simultaneous force and patch-clamp measurements on living cell tissues

Abstract

For the investigation of mechanosensitive ion channels of living cells it is of great interest to apply very local forces in the piconewton range and to measure, simultaneously, ion currents down to 1 pA. Scanning force microscopy (SFM) is a suitable technique, that allows the application of such small forces with a lateral resolution in the range of 10 nm. We developed a novel type of experimental setup, because no existing SFM, home built or commercial, allows a simultaneous investigation of ion currents and mechanical properties of living cells. The construction consists of a SFM that is combined with an upright infrared differential interference contrast (DIC) video microscope and a conventional patch-clamp setup. Instead of the object, the force sensor is scanned to prevent relative movements between the patch pipette and the patched cell. The deflection of the SFM cantilever is detected with the so-called optical deflection method through the objective of the optical microscope. In opposite to common optical setups the laser beam was not focused on the force sensor. The presented optic creates a parallel laser beam between the objective and the SFM cantilever, which allows a vertical displacement of the sensor without any changes of the detector signal. For the three-dimensional positioning of the specimen chamber a two-axis translation stage including a vertical piezoelectric translation device was developed. The SFM tip is fixed on a combined lateral and vertical translation stage including a piezoelectric tube scanner for three-dimensional fine positioning. Thus the instrument enables an easy approach of the SFM tip to any optically identified cell structure. The head stage of the patch-clamp electronics and the patch pipette are directly fixed on the specimen stage. This prevents relative movements between patched cells and patch pipette during the approach to the SFM tip. The three-axis positioning of the patch pipette is done by a compact hydraulic manipulator. With this combined setup, subcellular structures can be identified on living cells with the video microscope and simultaneously investigated with the SFM and the patch-clamp pipette. The features of the instrument are demonstrated with preparations of cultivated neuronal cells. Simultaneous measurements of ion current and force in organotypic cultures of mechanosensitive hair cells of the inner ear are proposed, as investigations of cell tissue preparations of up to 400 μm thickness are possible with this instrument.