High-Speed Hopping Probe Scanning Ion Conductance Microscopy

Abstract

Vertically protruding stereocilia on the apical surface of the inner ear hair cells represent the ultimate challenge for scanning probe imaging. We have previously imaged these structures, which are largely unresolvable by optical microscopy, using hopping probe scanning ion conductance microscopy (HPSICM) in glutaraldehyde-fixed mammalian auditory hair cells (Novak et al. Nat Methods, 2009). However, it required a considerable amount of time (∼44 min per bundle of ∼8x8 μm). To study live cells, we needed to significantly increase the speed of imaging.For Z-movement we have now used a faster piezo assembly with a resonant frequency of ∼18 kHz, similar to the ones we used for hair bundle deflection. Despite having a less sensitive strain gauge sensor, the vertical resolution of the system remained the same (∼5nm). After adjusting the proportional-integral-derivative controller of the Z-scanner (∼50μs delay) and increasing the speed of approach, we were able to obtain high-resolution images of live hair cell bundles at a frame rate of 12 min/bundle or less.We tested the performance of the improved HPSICM system in live rat inner hair cells (IHC) and showed, for the first time in live cells, the presence of characteristic stereocilia features at an X-Y resolution of ∼11nm. We also imaged IHC bundles from the Shaker2 and Whirler mice due to their short stereocilia with abundant stereocilia links (typically ∼5nm in diameter and ∼100-300nm in length). We confirmed the reproducibility of links in continuous time-lapse scanning and also their absence after chemical disruption with BAPTA-buffered Ca2+-free medium.Our results demonstrate that the improved HPSICM technique successfully visualizes the extremely convoluted surface of stereocilia in live auditory hair cells at a high resolution and a faster speed.Supported by NIDCD/NIH (R01DC008861).