Touching the cell without contact: a comprehensive review of scanning ion conductance microscopy methodologies for advanced subcellular mechanical imaging.

Scanning ion conductance microscopy (SICM) is a unique technique that enables non-contact, high-resolution, multiparametric imaging of living cells under physiological conditions. Recent advances have extended the capabilities of SICM to quantify local nanomechanical properties - fundamental regulators of cell function and state. Cellular stiffness serves as a sensitive biomarker of pathological processes, including cardiovascular, oncological, and neurodegenerative diseases. This review provides a systematic comparison of SICM-based methods for stiffness measurements, based on applied hydrostatic pressure, intrinsic colloidal forces, and electroosmotic flow pressure. For each approach, we critically evaluate implementation principles, advantages, and limitations, providing guidance for selecting the most suitable method accounting for experimental requirements and available instrumentation. By drawing on examples from cytoskeletal dynamics, cell migration, biomaterial-cell interactions, and pharmacological screening, this review highlights the transformative potential of nanomechanical mapping, positioning SICM as a uniquely versatile tool for investigating cellular properties and behavior in both health and disease.