Single-molecule imaging of stochastic signaling events in living cells.

Environmental changes result in signaling events at cell membranes. To develop the means to understand these events at the molecular level, it is essential to become familiar with the stochastic nature of signaling molecules in living cells. Using total internal reflection fluorescent microscopy (TIRFM), these signaling events can be directly observed at the single-molecule level. This article explains the basis of TIRFM and how it is set up. It then describes how to visualize cell membrane signaling events. It also explains how to prove that detected fluorescence is emitted from single dye molecules and how to analyze the data from TIRFM experiments.