Atomic Force Microscopy-Based Nanomechanical Profiling Reveals Oxidative Stress-Associated Structural Alterations and...

Chicken erythrocytes are nucleated blood cells that provide a useful model for assessing oxidative stress-related membrane injury, but the conventional hemolysis assays do not resolve single-cell nanoscale mechanical alterations. Here, we established an atomic force microscopy (AFM)-based workflow to characterize structural and nanomechanical alterations associated with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) exposure and curcumin-associated protection in chicken erythrocytes. Hemolysis assays were used to select a moderate injury condition and a protective curcumin dose. Confocal microscopy was used to assess filamentous actin (F-actin) organization and nuclear morphology, and short-term storage conditions were optimized to preserve fragile damaged cells before AFM measurements. The AAPH-induced concentration-dependent hemolysis, cytoskeletal disruption, and nuclear shrinkage altered erythrocyte morphology, characterized by decreased cellular dimensions and increased cell height. These treatments also affected nuclear mechanical properties, as indicated by increased indentation depth of the nuclear region and decreased apparent Young's modulus and adhesion force at the nuclear region. Storage in Alsever's solution at 4 °C best preserved the damaged erythrocytes for AFM analysis. Curcumin pretreatment reduced hemolysis and partially preserved cytoskeletal and nuclear morphology. AFM further showed that curcumin attenuated the nanoscale topographical and mechanical changes induced by AAPH. These results link biochemical injury, subcellular structural remodeling and single-cell nanomechanical phenotypes, and provide a quantitative AFM workflow for evaluating oxidative damage-associated structural and mechanical alterations and curcumin-associated preservation in fragile blood cells.