We previously imaged stereocilia links in glutaraldehyde-fixed mammalian auditory hair cells using hopping probe scanning ion conductance microcopy (HPSICM, Novak et al. Nat Methods, 2009). Due to the complex three-dimensional structure of the stereocilia bundle, achieving high-resolution images required a considerable amount of time (∼44 min/bundle). To study the dynamics of stereocilia link regeneration in real time, we needed a significantly faster yet reliable way to continuously scan the stereocilia bundles in live hair cells.To improve the HPSICM imaging speed, we re-designed the scan head by incorporating a faster piezo assembly with a resonant frequency of ∼18 kHz for Z-movement. Despite having a less sensitive strain gauge sensor (compared to the previously used capacitive sensor), the vertical resolution of the system remained the same (∼5nm). Significantly smaller inertia allowed mounting the scan head on a rotational platform and scanning the sample at any angle, a pre-requisite for the successful imaging of tip links. Moreover, the overall image resolution was slightly decreased and we can now image hair cell bundles significantly faster (∼11 min/bundle).The performance of the improved system was tested using cultured organ of Corti explants from the Shaker 2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). To test the ability of HPSICM to detect these miniature structures at high imaging speed we performed continuous time-lapse scanning and looked for reproducibility of the links in consecutive images. Next, to rule out the possibility that the observed links were simply scanning artifacts, we disrupted the links by treating the explants with BAPTA-buffered Ca2+-free medium. Our results demonstrate that the improved HPSICM technique successfully visualizes stereocilia links in live auditory hair cells.Supported by NIDCD/NIH (ARRA supplement to R01DC008861)