The atomic force microscope (AFM) was conceived to image surfaces of insulators with the atomic resolution that a scanning tunneling microscope (STM) had already provided for metal and semiconductor surfaces.  In the meantime, scanning probe methods have turned into prime tools for quantum physics and chemistry at surfaces and interfaces.  Besides the capability of modern instruments to design matter atom by atom, this stupendous development is due to the invention of a rapidly oscillating piezoceramic tuning fork as a force probe and the functionalization of the microscope tips with specific atoms or molecules.  These features were used to probe for the first time the local chemical reactivity within a single molecule in challenging experiments where the spatial separation of two atoms is reduced to less than a bonding length.

A single CO molecule that is transferred from a surface to the microscope tip applying known protocols acts as the sensor for chemical reactivity.  Figure 1 illustrates the procedure of picking up a single CO from the surface.  To this end, an ensemble of CO molecules is coadsorbed with the target molecule to be studied.  In STM images, a single CO molecule appears as a circular depression (arrow in Figure 1a).  Upon sufficiently close approach to an adsorbed CO the biased tip is decorated with the CO.  The resulting tip carries a single CO molecule where the O atom constitutes the outmost apex atom.  The successful transfer is proved by the henceforth missing CO on the surface and the increased spatial resolution of the target molecules (Figure 1b, Figure 1b*) as well as by the appearance of adsorbed CO as circular protrusions (Figure 1c).
 

This tip is now used to explore the local chemical reactivity of a simple dye.  In the present case, it is a phthalocyanine (2H-Pc, Figure 2a) that appears with a characteristic cloverlike shape in STM images (Figure 1, Figure 2a (left panel)).  The key experimental findings that support the idea of a single-molecule probe for intramolecular chemical reactivity are depicted in Figure 2b.  They show that the evolution of the short-range force between the CO probe and 2H-Pc depends on the actual approached intramolecular site.  The data sets reveal for two exemplary sites that the point of maximum attraction (force minimum marked (z*,F*)) exhibits a significantly stronger force F* at the macrocyclic center (red dot in the AFM image of Figure 2a) than at its periphery (blue).  The hypothesis that F* represents an indicator for the local chemical reactivity has been corroborated in additional experiments.
 

The macrocyclic center of 2H-Pc becomes more reactive upon removal of the pyrrolic H.  Indeed, it is known from conventional chemical experiments with a multitude of phthalocyanines that the encapsulation of a metal atom into the molecular center – the metalation reaction of the dye – evidences its increased chemical reactivity.  The H atoms can be removed in an atom-by-atom fashion using the scanning probe tip; that is, the chemical reaction 2H-Pc → H-Pc → Pc can be manually performed at the single-molecule level.   In that manner, the chemical reactivity of the center can be varied atom by atom.  Figure 3a shows an STM and AFM image of the resulting product molecule Pc.  As demonstrated in Figure 3b, the short-range force at the point of maximum attraction between CO and the macrocyclic center (bottom data set in Figure 3b) is indeed substantially stronger than observed from 2H-Pc.  The additional experiments corroborate that the magnitude of F* represents a viable quantitative measure for the local chemical reactivity.