Considerable recent progress has been made with respect to the generation, manipulation and characterization of molecules on surfaces using scanning probe microscopy (SPM). Such microscopy involves scanning an atomi- cally sharp tip across a surface to obtain an image. The tip serves another important purpose in that it can also be used for atomic manipulation. Various SPM instru- ments exist today. The year 1982 saw the invention of the scanning tunnelling microscope 1 , the development of which led G. Binnig and H. Rohrer to be awarded the Nobel Prize in Physics in 1986. The principles behind scanning tunnelling microscopy (STM) imaging involve electron tunnelling, a quantum mechanical effect. A complementary method, and one that this Review will focus on more, is atomic force microscopy (AFM), which was developed in 1986 by G. Binnig, C. F. Quate and Ch. Gerber 2 and led them to receive the Kavli Prize in Nanoscience in 2016. In AFM, the force acting between tip and sample is exploited to obtain images. In 1990, D. Eigler and E. K. Schweizer pioneered atomic manipulation, demonstrating the placement of atoms with atomic precision 3 using STM. Later mile- stones include reports by Sugimoto and co-workers describing atomic manipulation at room temperature 4 and demonstrating chemical sensitivity on atom inlays with AFM 5 . However, only few examples of molecu- lar reactions triggered by atomic manipulation have been reported until recently. Important works include the dissociation of dioxygen by Stipe and co-workers 6 , an Ullmann reaction induced by atomic manipula- tion by Hla and co-workers 7 and polymerization by Y. Okawa and M. Aono 8 , all performed and characterized using STM. At the time, a great challenge facing these scientists was the identification of the reaction products. The direct assignment of molecular structure by atomic -resolution imaging was yet to be enabled, and this problem led to the development of inventive methods to characterize reaction products. For example, the gen- eration of biphenyl from iodobenzene in the Ullmann reaction was confirmed by lateral manipulation: that is, pulling of the product 7 . Molecular orbital imaging by STM 9 increased our insight into molecular reactions and, for example, allowed observation of metal compl- exation 10 and tautomerization reactions 11 triggered by atom manipulation. It was in 2009 that AFM tip functionalization by atom manipulation allowed for molecular imaging at atomic resolution 12 . Subsequently, it became possible to identify even complex molecules from their images in real space 13 and to study reaction products by AFM. Concurrently, on-surface synthesis of covalently bonded molecules became an important topic of research 14 , and molecular wires and graphene nanoribbons 15 in par- ticular are now grown by on-surface synthesis, which involves thermal annealing of custom-made precursor molecules. Naturally, atomic-resolution AFM became an important tool to characterize the products and inter- mediates of thermally induced on-surface reactions 16–22 . In addition to product identification, AFM studies also afford a detailed understanding of reaction mecha- nisms, such that product selectivities and yields can be improved and new structures discovered. IBM Research – Zurich, Säumerstrasse 4, 8803 Rüschlikon, Switzerland. Correspondence to L.G. lgr@zurich.ibm.com doi:10.1038/s41570-016-0005 Published online 11 Jan 2017 Generation, manipulation and characterization of molecules by atomic force microscopy Niko Pavlic ˇek and Leo Gross Abstract | Using atomic manipulation, one can dissociate, form and rearrange bonds, as well as alter the conformation or charge state of molecules. The molecular structures of reactants, intermediates and products are revealed at unprecedented resolution by using atomic force microscopy (AFM) and a suitably functionalized tip. Our present capabilities of manipulation and imaging of molecules by AFM approach the level of control predicted by Richard P. Feynman in his famous lecture ‘There’s plenty of room at the bottom’, in which he described how molecules and materials might be formed by attaching and detaching individual atoms at will. In this Review, we discuss recent progress and the future prospects of molecule generation by atom manipulation and molecular characterization by AFM. REVIEWS NATURE REVIEWS | CHEMISTRY VOLUME 1 | ARTICLE NUMBER 0005 | 1 ©2017MacmillanPublishersLimited,partofSpringerNature.Allrightsreserved.