Quantification of cell membrane insertion events using force spectroscopy

Abstract

This study investigates the processes of cell membrane puncturing and the factors influencing these processes through force spectroscopy experiments. A custom-designed tall and sharp tip was fabricated on a commercially available microcantilever using two-photon polymerization (2PP). Force spectroscopy curves were measured at several locations on the cell while the tip was inserted inside the cell. A cell membrane insertion event, identified as a ”force drop” in force spectroscopy curves, is often hardly visible or does not occur. In this study, the effects of probe tip diameter, cantilever spring constant, insertion velocity, and cell height of mouse preosteoblast cells were systematically analysed for their influence on how often the force drops occur and how clearly they are visible. Data processing algorithms were employed to process thousands of force spectroscopy curves, enabling large sample statistical analysis using multiple linear regression. The results demonstrate that selecting cells with maximum height and size increases force drop occurrence from 10% to 40%. Furthermore, while decreasing the tip diameter from 2 to 0.7 µm did not affect the occurrence rate, it increased force drop visibility from ∼250 to ∼600 pico-Newton (pN). Additionally, cantilevers with a spring constant of 0.2 N/m achieved higher force drop occurrence (40%) and visibility (∼300 pN), compared to more flexible cantilevers with a spring constant of 0.02 N/m (20% occurrence, ∼40 pN visibility). These findings provide valuable insights for optimizing experimental parameters in cell membrane mechanics research.