Active Force Dynamics in Red Blood Cells Under Non-Invasive Optical Tweezers.

Red blood cells (RBCs) sustain mechanical stresses associated with microcirculatory flow through ATP-driven plasma membrane flickering. This is an active phenomenon driven by motor proteins that regulate interactions between the spectrin cytoskeleton and the lipid bilayer; it is manifested in RBC shape fluctuations reflecting the cell's mechanical and metabolic state. Yet, direct quantification of the forces and energetic costs underlying this non-equilibrium behavior remains challenging due to the invasiveness of existing techniques. Here, a minimally invasive method that combines bead-free, low-power optical tweezers with high-speed video microscopy is employed to track local membrane forces and displacements in single RBCs during the same time window. This independent dual-channel measurement enables the construction of a mechano-dynamic phase space for RBCs under different chemical treatments, which allows for differentiating between metabolic and structural states based on their fluctuation-force signatures. Quantification of mechanical work during flickering demonstrates that membrane softening enhances fluctuations while elevating energy dissipation. The proposed optical tweezers methodology provides a robust framework for mapping the active mechanics of living cells, enabling precise probing of cellular physiology and detection of biomechanical dysfunction in diseases.