Scanning ion-conductance microscopy with a double-barreled nanopipette for topographic imaging of charged chromosomes.

Abstract

Scanning ion conductance microscopy (SICM) is useful for imaging soft and fragile biological samples in liquids because it probes the samples' surface topography by detecting ion currents under non-contact and force-free conditions. SICM acquires the surface topographical height by detecting the ion current reduction that occurs when an electrolyte-filled glass nanopipette approaches the sample surface. However, most biological materials have electrically charged surfaces in liquid environments, which sometimes affect the behavior of the ion currents detected by SICM and, especially, make topography measurements difficult. For measuring such charged samples, we propose a novel imaging method that uses a double-barrel nanopipette as an SICM probe. The ion current between the two apertures of the nanopipette desensitizes the surface charge effect on imaging. In this study, metaphase chromosomes of Indian muntjac were imaged by this technique because, owing to their strongly negatively charged surfaces in phosphate-buffered saline, it is difficult to obtain the topography of the chromosomes by the conventional SICM with a single-aperture nanopipette. Using the proposed method with a double-barrel nanopipette, the surfaces of the chromosomes were successfully measured, without any surface charge confounder. Since the detailed imaging of sample topography can be performed in physiological liquid conditions regardless of the sample charge, it is expected to be used for analyzing the high-order structure of chromosomes in relation to their dynamic changes in the cell division.