We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by the AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample, but true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been reported. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, which allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . Until now, SICM has been restricted to imaging relatively flat surfaces, as all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM, a nanopipette is mounted on a three-dimensional piezoelectric translation stage and automatic feedback control moves the pipette up or down to keep the pipette current constant (the set point) while the sample is scanned in x and y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features We illustrate the benefits of HPICM in In contrast to conventional raster scanning, HPICM has the additional advantage that the order of imaging pixels is not predetermined. Therefore, we divided the entire image into equal-sized square
