Scanning probe microscopy (SPM) uses a probing tip which scans over a sample surface for obtaining information on the sample surface characteristics. Among various types of SPM, atomic force microscopy (AFM) has been widely applied to imaging of biological samples including chromosomes. Scanning ion conductance microscopy (SICM) has been also introduced for visualizing the surface structure of biological samples because it can obtain "contact-free" topographic images in liquid conditions by detecting ion current flow through a pipette opening. However, we recently noticed that the consistent imaging of chromosomes is difficult by SICM. In this paper, the behaviors of the ion current on the sample surfaces were precisely investigated for obtaining SICM images of isolated muntjac metaphase chromosomes more consistently than at present. The present study revealed that application of positive potential to the pipette electrode was acceptable for obtaining the topographic image of chromosomes, while application of negative potential failed in imaging. The approach curves were then studied for analyzing the relationship between the ion current and the tip sample distance when the pipette is approaching chromosomes. The current-voltage (I-V) curve further provided us the accurate interpretation of the ion current behavior during chromosome imaging. These data were further compared with those for SICM imaging of HeLa cells. Our findings indicated that chromosomes are electrically charged and the net charge is strongly negative in normal Dulbecco's phosphate buffered saline. We finally showed that the ion concentration of the bath electrolyte is important for imaging chromosomes by SICM.
Keywords: approach curve; biological imaging; charged samples; chromosomes; current-voltage curve; scanning ion conductance microscopy.