Adaptive Hopping Probe Ion Conductance Microscopy of Live Cells at ∼5-10 NM Resolution

The inherent low imaging speed of hopping probe ion conductance microscopy (HPICM) poses a constraint for the study of living cells at super resolution. The duration of each imaging frame further increases when the cell topography requires large approach/retraction movements of the scanning pipette. We have modified our HPICM system to increase its imaging speed and sensitivity.We replaced the Z-scanner with a ∼18 kHz resonant frequency piezo assembly to allow for faster pipette movements. An external proportional-integral-derivative controller was used to decrease the delay in the Z-scanner. In our modified HPICM algorithm the approach speed adapts to the changes in the ionic current flowing through the pipette. Altogether, our changes to the HPICM system allowed for approach speeds up to 4X faster with delays of only ∼50μs. Our “adaptive” approach curve effectively reduced the noise floor by half, which improved the vertical resolution of the system to ∼5nm. We also introduced adaptive filtering of the ionic current, which decreased by half the minimum setpoint, thus improving the system's sensitivity.To test the performance of the improved HPICM setup, we imaged the vertically protruding stereocilia (∼0.5-3μm in height, ∼100-400nm in diameter) of the rat inner ear sensory cells. When we previously imaged these structures with HPICM, we used glutaraldehyde-fixed cells and each imaging frame took >44 min (Novak et al. Nat Methods, 2009). Now, the entire stereocilia bundle (∼9x9μm) in a live cell is imaged in ∼15 min and sub-regions of interest (∼2x2μm) in ∼3-6 min, with ∼10-30nm XY resolution, even with the hop amplitudes of 3-5μm.Our improved HPICM system allows for the faster imaging of live cells at nanometer resolution, even in the cells with very convoluted topographies.Supported by NIDCD/NIH (R01DC008861, R01DC014658).