AbstractThe combination of atomic force microscopy (AFM) with nanofluidics, also referred to as FluidFM, has facilitated new applications in scanning ion conductance microscopy, direct force measurements, lithography, or controlled nanoparticle deposition. An essential element of this new type of AFMs is its cantilever, which bears an internal micro-channel with a defined aperture at the end. Here, we present a new approach for in-situ characterization of the internal micro-channels, which is non-destructive and based on electrochemical methods. It allows for probing the internal environment of a micro-channeled cantilever and the corresponding aperture, respectively. Acquiring the streaming current in the micro-channel allows to determine not only the state of the aperture over a wide range of ionic strengths but also the surface chemistry of the cantilever’s internal channel. The high practical applicability of this method is demonstrated by detecting the aspiration of polymeric, inorganic and hydrogel particles with diameters ranging from several µm down to 300 nm. By verifying in-situ the state of the aperture, i.e. open versus closed, electrophysiological or nano-deposition experiments will be significantly facilitated. Moreover, our approach is of high significance for direct force measurements by the FluidFM-technique and sub-micron colloidal probes.