The invention of scanning force microscopy (SFM) (1) and its modification to optical detection of forces (2) has opened the exciting perspective of imaging the surface of living biological specimens (3)(4)(5). The additional potential of SFM for the study of molecular recognition, using a measuring tip with ligands bound, has recently gained much attention. The idea is to detect and study the binding of ligands on tips to surface-bound receptors by applying an increasing force to the complex that reduces its lifetime until it dissociates at a measurable unbinding force. So far, interaction forces were reported for the ligand-receptor pair biotin-avidin (6-8) and for complementary DNA nucleotides (9, 10). For these studies, SFM tips were covered with immobilized ligands. This strategy failed for antibody-antigen recognition (11), and the failure was attributed to the lack of molecular mobility and to unspecific tip-probe adhesion forces, obscuring specific interactions. Apart from detection and study of single recognition events, the concept of using SFM tips with ligands ("sensors") has further perspectives: (i) for

Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.

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Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibodyantigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.