Abstract
Erythrocyte (RBC) aging involves significant structural and nanomechanical alterations crucial to their function. This study aims to bridge the gap between analyses based on statistical morphometric parameters, e.g., membrane roughness, and those based on point-dependent nanomechanical properties, e.g., stiffness or Young's modulus. Using Atomic Force Microscopy, we investigated morphology, membrane roughness, and nanomechanical properties on the very same RBCs under dehydrated (air) and hydrated (physiological buffer) conditions. The cells were studied at different stages of in vitro aging: one, seven, and 12 days. Our results quantitatively show that across dehydration, as well as along the aging pathway, RBCs become progressively more rigid while their membrane roughness decreases, a trend observed in both environments. Notably, the differences between the hydrated and dehydrated states were large in young cells but diminished when erythrocytes aged. Despite these parallel trends, high-resolution mapping on the nanoscale revealed that roughness and Young's modulus do not correlate, indicating that these parameters are linked to different properties. In conclusion, this work provides a comprehensive protocol for a biophysical description of RBC aging and establishes that the simultaneous measurement of membrane roughness and nanomechanical properties offers a complementary approach, yielding a more complete characterization of cellular properties.