Focal adhesions play an important role in cell spreading,migration,and overall mechanical integrity.The relationship of cell structural and mechanical properties was investigated in the context of focal adhesion processes.Combined atomic force microscopy(AFM) and laser scanning confocal microscopy(LSCM) was utilized to measure single cell mechanics,in correlation with cellular morphology and membrane structures at a nanometer scale.Characteristic stages of focal adhesion were verified via confocal fluorescent studies,which confirmed three representative F-actin assemblies,actin dot,filaments network,and long and aligned fibrous bundles at cytoskeleton.Force-deformation profiles of living cells were measured at the single cell level,and displayed as a function of height deformation,relative height deformation and relative volume deformation.As focal adhesion progresses,single cell compression profiles indicate that both membrane and cytoskeleton stiffen,while spreading increases especially from focal complex to focal adhesion.Correspondingly,AFM imaging reveals morphological geometries of spherical cap,spreading with polygon boundaries,and elongated or polarized spreading.Membrane features are dominated by protrusions of 41-207 nm tall,short rods with 1-6 μm in length and 10.2-80.0 nm in height,and long fibrous features of 31-246 nm tall,respectively.The protrusion is attributed to local membrane folding,and the rod and fibrous features are consistent with bilayer decorating over the F-actin assemblies.Taken collectively,the reassembly of F-actin during focal adhesion formation is most likely responsible for the changes in cellular mechanics,spreading morphology,and membrane structural features. Focal adhesions play an important role in cell spreading, migration, and overall mechanical integrity. The relationship of cell structural and mechanical properties was investigated in the context of focal adhesion processes. Combined atomic force microscopy (AFM) and laser scanning confocal microscopy (LSCM) was utilized to measure single cell mechanics, in correlation with cellular morphology and membrane structures at a nanometer scale. Characteristic stages of focal adhesion were verified via confocal fluorescent studies, which confirmed three representative F-actin assemblies, actin dots, filaments networks, and long and aligned fibrous bundles at cytoskeleton. Force-deformation profiles of living cells were measured at the single cell level, and displayed as a function of height deformation, relative height deformation and relative volume deformation. As single adhesion cell profiles both membrane and cytoskeleton stiffen, while spreading greater especia lly from focal complex to focal adhesion. Correspondingly, AFM imaging reveals morphological geometries of spherical cap, spreading with polygon boundaries, and elongated or polarized spreading. Membrane features are dominated by protrusions of 41-207 nm tall, short rods with 1-6 μm in length and 10.2-80.0 nm in height, and long fibrous features of 31-246 nm tall, respectively. protrusion is attributed to local membrane folding, and the rod and fibrous features are consistent with bilayer decorating over the F-actin assemblies. Taken collectively, the reassembly of F-actin during focal adhesion formation is likely likely for the changes in cellular mechanics, spreading morphology, and membrane structural features.