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

The lateral mobility of lipids in phospholipid membranes has attracted numerous experimental and theoretical studies, inspired by the model of Singer and Nicholson (1972. Science, 175:720-731) and the theoretical description by Saffman and Delbrück (1975. Proc. Natl. Acad. Sci. USA. 72:3111-3113). Fluorescence recovery after photobleaching (FRAP) is used as the standard experimental technique for the study of lateral mobility, yielding an ensemble-averaged diffusion constant. Single-particle tracking (SPT) and the recently developed single-molecule imaging techniques now give access to data on individual displacements of molecules, which can be used for characterization of the mobility in a membrane. Here we present a new type of analysis for tracking data by making use of the probability distribution of square displacements. The potential of this new type of analysis is shown for single-molecule imaging, which was employed to follow the motion of individual fluorescence-labeled lipids in two systems: a fluid-supported phospholipid membrane and a solid polymerstabilized phospholipid monolayer. In the fluid membrane, a high-mobility component characterized by a diffusion constant of 4.4 microns2/s and a low-mobility component characterized by a diffusion constant of 0.07 micron2/s were identified. It is proposed that the latter characterizes the so-called immobile fraction often found in FRAP experiments. In the polymer-stabilized system, diffusion restricted to corrals of 140 nm was directly visualized. Both examples show the potentials of such detailed analysis in combination with single-molecule techniques: with minimal interference with the native structure, inhomogeneities of membrane mobility can be resolved with a spatial resolution of 100 nm, well below the diffraction limit.

In recent years observations at the level of individual atoms and molecules became possible by micros

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