Visualizing single molecules in living Dictyostelium cells using total internal reflection fluorescent microscopy (TIRFM)

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 protocol describes the procedure used for objective-type TIRFM to visualize single fluorescent molecules in living Dictyostelium cells, which are highly sensitive to chemoattractant stimulation. This method can also be applied to other cell types.