Single-molecule approaches. (A) AFM. A tip is attached to a cantilever, with deflection of the tip or changes in its resonance frequency reporting on proximity to features on a cellular surface. By raster scanning the sample, an image of the 3D shape can be formed with subnanometer resolution. (B) OT. A functionalized bead is introduced into the cell. The bead is trapped and manipulated by a focused laser beam. (C) MT. Magnetic beads that specifically interact with a substrate of interest are introduced into the cell. By applying a magnetic field, the beads can be rotated or translated, thereby introducing a force to the system. (D) Fluorescence microscopy. Substrates of interest are labeled with a fluorescent tag. Their fluorescence is detected on a sensitive camera, allowing real-time visualization of spatiotemporal dynamics. (E) PAINT. This technique works by labeling a substrate that interacts transiently with a receptor. A low concentration of fluorescent ligands is introduced in the extracellular medium such that at a constant rate, receptors in the membrane are being visualized by short-lived fluorophore immobilization during the imaging sequence. (F and G) smFRET. (F) Two substrates of interest are labeled with two specific fluorescent tags (a donor–acceptor FRET pair). The emission of the donor tag spectrally overlaps with the absorption of the acceptor dye. The donor transfers its energy to the acceptor in a distance-dependent manner (FRET). An interaction between the two substrates will give a FRET signal, providing a dynamic observation of molecular interactions. (G) A molecule of interest is labeled with a FRET pair at known positions, one with a donor and the other with an acceptor. A change in the conformation of the substrate can be observed as a change in the FRET efficiency.