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

ABSTRACT 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.