Integrated SICM-AFM-optical microscope to measure forces due to hydrostatic pressure applied to a pipette

Scanning ion conductance microscopy (SICM) is a scanning probe microscopy particularly suitable for the investigation of living biological specimens due to its low invasivity. Recently, this technique has been used not only to perform 3D-imaging, but also to stimulate and guide neuronal growth cones. In particular, it has been demonstrated that one can guide the cone growth for tens of micrometres by means of recurrent and non-contact SICM scanning along a defined line, with a pipette having an internal hydrostatic pressure. Accurate measurements of the mechanical forces acting on the cell membrane in these stimulation protocols are essential to explain the biological mechanisms involved. Herein a setup specifically developed for this purpose, combining together SICM, atomic force microscopy (AFM) and inverted optical microscopy is described. In this configuration, a SICM pipette can be approached to an AFM cantilever while monitoring the cantilever deflec- tion as a function of the hydrostatic pressure applied to the pipette and the relative distance. In this way, one can directly measure mechanical forces down to 20 pN. The same apparatus is thus sufficient to calibrate a given pipette and immediately use it to study the hydrostatic pressure effects on living cells.