The effect of the thickness of ultrathin sections on the topographical contrast in the images of atomic force microscopy (AFM) had been studied. Three different cell lines, Tca8113, C6 and ECV-304 were treated with conventional TEM fixation and embedment techniques. Cut by ultramicrotomy and collected with mica pieces, thin sections with different thickness were imaged by AFM on both sides. The images observed from the lower surfaces showed that the cell regions were always concave compared to the epoxy resin regions, and the concavity was increased with the increase of the ultrathin section thickness. Interestingly, the images from upper surfaces showed a peculiar convexness in the cell regions when the thickness is small, and achieved a maximum when the thickness was about 80 nm, and finally became concave when the thickness was larger than 120 nm. Statistic analysis showed that this trend was a general phenomenon. The relevant mechanism has been discussed.