a, b, Average tip motions observed at different A ratio in buffer solution during HS-AFM imaging on mica (a) and a supported lipid bilayer (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DOPS, 4:1) (b). c, d, Forces caused by the elastic response of the cantilever (top), the hydrodynamic damping with the medium (middle) and the total force that governs the tip motion (bottom). e, f, Sum of the drive force and tip-sample interaction force. g, h, Reconstructed F ts trajectories during a single oscillation cycle, based on the point-mass model (equation (7), equation (8), Methods). i, Comparison between peak forces obtained from reconstructed F ts trajectories on mica (blue dashed line) and membrane (red dashed line), and peak forces simulated using different surface stiffness using VEDA38 (dashed lines and grey shadowed area). The VEDA simulation is performed by using the amplitude-modulation-approach curve tool with the following settings: discrete approach steps within a defined z-range, acoustic excitation, A free = 2 nm, Hertz contact model (E tip = 130 GPa, ν tip = 0.3, ν sample = 0.5, ν is the Poisson’s ratio) with a tip radius of 1 nm, and other HS-AFM experimental parameters (for example, k, Q and ω 0 ). j, Comparison between average forces obtained from reconstructed F ts trajectories on mica (blue dashed line) and membrane (red dashed line) and values calculated through equation (1) (thick black dashed line). k, Comparison between peak forces obtained from experimental F ts trajectories on membrane at different A free values. Using second-order polynomial fitting, the peak force reconstructed in the condition of A free = 1.5 nm can be well-described by y = −688.7x2 + 633.4x + 55 (black dashed line) with R2 = 0.99. This fitting allows us to estimate the upper bound of force (peak force) applied to PIEZO1 channels at any given A ratio . Tip trajectories are representative of ≥5 independent experiments using ≥3 different HS-AFM cantilevers.