A team of experts in the fields of biophysics, engineering, nanotechnology and computational biology was formed. The team developed two new nanotools to probe adhesion and mechanics on living cells: 1) high-speed atomic force microscopy (HS-AFM) coupled to fast confocal fluorescence microscopy that gives access to force measurements at the shortest timescales (sub-microsecond), and 2) acoustic force spectroscopy (AFS) coupled to reflection interference contrast microscopy (RICM) that gives access to biomechanical processes at long timescales (hours). The new nanotools were calibrated on model systems, and adapted and improved to work on living cells. We developed dedicated control software for the two instruments and to synchronize them with the coupled optical microscopes. We developed associated data analysis software that allows fast and robust extraction of the mechanical parameters from experimental data. We applied the system to living cells revealing the mechanics and adhesion of white blood cells and viruses. We found that white blood cells become stiffer and stickier under conditions under inflammatory conditions. The technology developed allowed us to study the first step of SARS-CoV-2 virus infection, by deciphering the mechanisms of the spike protein binding to its receptor. The AFS development allowed us to probe the interaction between receptor and ligands at ultraslow loading rates. Combined with our HS-AFM system and simulations, we cover an unprecedented total range expanding 15 orders of magnitude in time. This opens the door to unexplored biophysical regimes.
We have published our results through various works in peer reviewed journals (Karageorgi et al. 2020; Ilić et al. 2022; Martins et al. 2022; Junior et al. 2023; Eroles, et al. 2023; Wang et al. 2023; Mesbah et al. 2024; Saha et al. 2024). Two methodological works on AFM calibration methods, necessary for robust and quantitative measurements (Sumbul et al. 2020; Rodriguez-Ramos and Rico, 2021). Five review articles on HS-AFM, AFM instrumnetation, cell mechanics, and mechanical biomarkers (Valotteau et al. 2019; Casuso et al 2020; Lacaria et al. 2023; Casuso et al. 2023; Eroles and Rico 2023). We have disseminated the developed software as open-source trhough repositories for being used and improved by the research community (Alonso et al. 2023).
