Besides imaging, AFM may also be used as a force detector, directly obtaining information on the localization, adhesion, elasticity, and interaction of molecules (Hinterdorfer and Dufrene
2006; Muller
et al.
2009). In the force spectroscopy mode, interaction forces are measured by recording the deflection of the AFM tip cantilever while the tip moved up and down, thus yielding a force-distance curve (Oesterhelt
et al.
1999). Furthermore, acquiring force-distance curve makes it possible to map interactions between molecule and molecule/cell (Gaboriaud
et al.
2008). Mapping and functionally analyzing single molecules using so-called single-molecule force spectroscopy (SMFS) requires modification of the AFM tip with specific ligands, like antibodies or lectins, and then measuring the specific interaction force between the ligand and its receptor (Strunz
et al.
1999). Functionalization of the AFM tip is achieved using cross-linker molecules that anchor ligands firmly at low density, while maintaining their mobility and functionality (Barattin and Voyer
2008). SMFS can help us understand cell surface proteins such as adhesions assemble into nanodomains on the surface of living cells (Alsteens
et al.
2010; Dupres
et al.
2005). SMFS may also be exploited to pull on single molecules in order to study their elasticity, a property that plays an important role in cell behavior (Dupres
et al.
2009). To understand the dynamic process during cell entry in living cells and avoid disturbing AFM tip cantilever moving, a technique based on SMFS, force tracing, was developed by Wang
et al. (Pan
et al.
2015). During the force tracing test, the AFM tip cantilever bends downward, which causes a transient deflection; subsequently, the feedback system adjusts the position of the piezoelectric ceramic to maintain the deflection of the cantilever, which could be converted to force (Pan
et al.
2015). The change of position during substance entry cell can also be monitored using constant force mode. The performing details of the force tracing technique are described as following: (1) Before performing the force tracing test, the AFM tip connected with the target substance is located above the relatively flat region of cell periphery with the help of CCD observation; (2) The target substance modified AFM tip moves toward the cell surface and contacts the living cell at a constant force/position through the fine-tuning of the feedback system, then the feedback system is closed; (3) In constant position mode, the force and duration of substance attached on AFM tip entry into cells will be detected by recording the deflection of the AFM tip cantilever and collecting by a PCI data acquisition card (Pan
et al.
2017). The force tracing shows unique advantages for detecting the force, duration, and displacement during the dynamic membrane transporting process at single molecules/particles level in living cells.