The structure of human fibroblasts has been characterised in vitro by atomic force microscopy (AFM) operated in the imaging or in the force vs. distance (F–d) modes. The choice of growth substrate is important to ensure good adhesion. The substrate also affects the imaging conditions for in vitro analysis of live cells; activated tissue culture dishes are shown to promote conditions that routinely result in good quality images. A qualitative model suggests that the activated substrate may act as a preferential scavenger of cellular debris, therefore promoting low adhesion between tip and membrane and preventing the tip from biofouling. Alternatively, the activated substrate may promote a more rigid cell structure, thus resulting in improved imaging. Good imaging conditions provide nondestructive in vitro information about cytoskeletal structure and dynamics; thus, treatment with cytochalasin can be monitored in real time for durations of several hours.