Hopping-Mode Scanning Ion-Conductance Microscopy Resolution during Synaptic Imaging

Abstract

Scanning ion conductance microscopy (SICM) is a non-contact scanning-probe topography imaging technique, in which current measured between an electrolyte-filled nanopipette and an external bath electrode is monitored through closed-loop feedback to maintain a constant distance between the probe and the sample. Topography is mapped by scanning this nanopipette laterally across the sample in either direct-current or alternating-current mode. Both modes suffer from limitations when imaging in the presence of large morphological undulations, such as with neuronal bodies. The hopping-probe scanning (HP-SICM) mode, however, is ideally suited for imaging neuronal topography. In HP-SICM, the pipette current reduces upon approaching the sample, and height is recorded when the current reaches a pre-determined threshold (i.e. a fraction of the far-away value). The probe then retracts by a distance larger than the tallest structural feature, moves laterally by a pre-defined step, and reapproaches the sample. Here, using detailed finite-element simulations incorporating the Poisson-Boltzmann and Nernst-Planck formalisms, we examine the factors that affect HP-SICM resolution when imaging synapses in live hippocampal cultures. We find that currents originating from the imaged membrane can affect the recorded sample height, thereby introducing artifacts that can range from a few nm to ∼100nm. This feature of HP-SICM resolution critically depends on the set threshold value, pipette concentration and pipette geometry but provides an opportunity to extracellularly map activity from small dendritic spines and pre-synaptic terminals (diameter < .5um) that are practically inaccessible with conventional electrophysiology. Last, we explore the current-clamp response near the membrane and find that pipette geometry and local ion gradients affect the voltage response. Funding: U. S. Army Research Laboratory and U. S. Army Research Office. Contract number W911NF-12-1-0594 (MURI).