SICM measures surface topography by detecting ion current changes through a nanopipette in a liquid environment, making it particularly suitable for imaging delicate biological samples in their native, liquid conditions without physical contact.

SICM is a non-contact technique that precisely measures surface topography by monitoring the ionic current flowing between an electrolyte-filled nanopipette (inner diameter typically ~100 nm) and a bath electrode in solution. The pipette tip is positioned close to the sample surface, which is also immersed in the same electrolyte. As the tip approaches the surface, the confined space between the tip and the sample restricts the movement of ions, causing a measurable decrease in the ion current (the ion current flow is analogous to an electrical current flowing through a variable resistor). This change in current is detected by a current amplifier connected to the pipette, enabling the system to maintain a constant current—and therefore a constant tip-to-sample distance—without physical contact.

When the nanopipette is far from the sample surface (a), ions can flow freely through the pipette opening, resulting in a high ion current. As the pipette approaches the surface (b), the space for ion flow becomes restricted, and the ion current decreases sharply. By setting a specific ion current value as a setpoint, the system can maintain this constant current at each measurement location by adjusting the pipette height. The plot shows how ion current drops as the pipette approaches the surface, illustrating the sharp change used to control height during scanning.

This mechanism demonstrates that SICM achieves surface detection and topographic imaging with true non-contact operation by employing distance-dependent ion current feedback to precisely control the pipette-sample separation. As a result, SICM is particularly well-suited for mapping the morphology of soft, fragile, or highly sensitive biological and polymer samples in liquid, as it eliminates mechanical stress and minimizes sample perturbation under physiologically relevant conditions.

SICM was employed to compare the nanoscale surface morphology of untreated Huh7 cells with those exposed to the non-ionic detergent Triton X-100. The untreated cells exhibited a smooth and clean surface, indicating an intact cell structure. Conversely, detergent treatment caused significant surface deterioration, characterized by the formation of tiny holes and disruptive surface structures. Critically, this non-invasive SICM imaging provided clear results into the structural changes of the cell membrane induced by detergent interaction, enabling the successful visualization of these effects on even very delicate and complicated biological samples without inducing mechanical stress or deformation.

This long-term live cell study observed two adjacent cells in a nutrient-deficient medium (1:9 cell culture medium to PBS ratio). SICM imaging captured changes over 152 minutes. Initially connected, the cells' edges began to separate after 30 minutes, resulting in complete disconnection. While cells normally form stable bonds, the harsh environment led to competition for resources, slowing growth and viability. This lack of nourishment and essential factors prevented the cells from maintaining the necessary adhesion molecules and signaling cues to stay attached to each other and the surface.