Probing true and apparent chemical bonds by atomic force microscopy

High-resolution atomic force microscopy (HR-AFM) images often show bond-like features between atoms, but when these features reflect true chemical bonds remains unclear. Here, we report a quantitative benchmark showing that, with a CO-functionalized tip, apparent bond-like features appear when the interatomic separation falls below a critical upper limit of 4.4–5.1 Å. This threshold holds across non-bonded CO clusters, an ionic NaCl(100) surface, and a hydrogen-bonded H 2 O hexamer. True bonds are detected only when their electron density is comparable to or greater than that above adjacent atoms, as shown in tolane. We further propose practical criteria to identify true bonds: flat profiles or local maxima at bond centers signify true bonds, whereas local minima indicate a steric origin, and overlap of the tip's van der Waals surface with that of the bond confirms a bond contribution. These results provide a reliable framework for interpreting HR-AFM images.