Tunable Single-Cell Multistate Nanomechanical Phenotyping by Micropipette-Assisted Atomic Force Microscopy to Dissect Cellular Heterogeneity

Mechanical forces are crucial for cellular function and disease, and particularly, atomic force microscopy (AFM)-based force spectroscopy has become a standard and important platform for characterizing the mechanical properties of single cells. Here, we present a study of micropipette-assisted AFM that enables multistate nanomechanical phenotyping of a living cell during its biological processes. Micropipette-assisted AFM offers the additional capability to manipulate single living cells in three dimensions, allowing the utilization of an AFM-based force spectroscopy assay to construct the dynamic nanomechanical phenotypes of single cells at multiple states. With micropipette manipulations, individual living cells could be selectively isolated in situ and subsequently positioned at specific locations on the engineered substrates with controllable properties. Subsequently, the mechanical changes of the same cells in the changed physiological states due to the interactions between cells and their altered microenvironments could be measured by AFM. The effectiveness of the proposed method was verified in a variety of systems, including single-cell responses to ECM biochemical cues, single-cell responses to ECM physical cues, and single-cell mechanics involved in cell–cell interactions within physical confinement, revealing numerous distinctive behaviors and nanomechanical phenotypes of individual cells. The study demonstrates an experimental approach to build the mechanical atlas of single cells undergoing regulated physiological and pathological changes, which offers additional possibilities for dissecting cellular heterogeneity from the biomechanical perspective and will benefit mechanobiology.