Establishing The Platform Of Interfacing 3D-MTC And STED Nanoscopy To Study Cell Biomechanics

Increasing evidence demonstrates that mechanical forces play cr itical roles in normal functions of living cells,tissues,and organisms and in diseases.Traditional biochemistry and molecular biology focus on how environment influences the human body and cells via the chemical and molecular signal pathways.These studies have made great progress but there are still many problems that cannot be answered.Biomechanics uses mechanical principles and methods to quantify relationships between structure and function of living cells and the human body.Applying mechanical forces on living cells by devices to simulate physiological mechanical environment of living tissues is a useful approach in the biomechanical study.However,currently no method is available that is able to apply mechanical forces to a living cell in any des ired direction,with varying frequency and duration and can quantify intracellular and intranuclear structural changes in real time with high spatial resolution that breaks the spatial resolution limit of light microscopy.Here we present a platform of interfacing 3D-Magnetic Twisting Cytometry(3D-MTC)with stimulated emission depletion(STED)nanoscopy.3D-MTC is a technique for applying mechanical stresses on a living cell by first magnetizing ferromagnetic beads bound to the cell surface in any desired direction and then exerting shear forces by generating magnetic twisting fields to rotate ferromagnetic beads in any other directions.STED nanoscope breaks the limit of optical diffraction by a physical method and obtains super spatial resolution of fluorescent molecules.The interfaced system connects 3D-MTC with STED nanoscope to synchronize magnetic field-induced mechanical forces to quantify nanometer-scale force-induced displacements with the super-resolved fluorescence STED microscopy enabled nanosc ale structural imaging.This integrated platform allows for rapid real time acquisition of a living cell's mechanical responses to local mechanical stresses via specific receptors and for quantifying intracellular structural changes in the same cell.We test the platform by measuring the cellular biomechanical properties,including cell stiffness,displacements and strains of nulcear proteins.External forces are applied from different directions on the cell surface,when the magnitude and direction of the applied forces change,the displacements and strains of the nuclear proteins are drastically different.This platform is a real-time synchronization,high resolution,accurate and convenient tool for measuring mechanical properties of cells,it will be powerful in cellular biomechanics experiments and quantitative analysis.This platform has been tested for its stability,reliability,and utility in living cells and is a significant tool box that thus will possibly uncover new horizons in cell mechanobiology.