Fig. 4.

Total internal reflection fluorescence (TIRF) microscopy for monitoring GPCR dimerisation. Unlike epifluorescence in which the laser is directed perpendicular to the coverslip, causing illumination of the entire sample volume, TIRF directs the laser at angle θ, resulting in illumination of only approximately 100 nm of the sample. When combined with FRET, this ensures that only molecules close to the coverslip are excited (red asterisk), as opposed to in epifluorescence when both intracellular and cell-surface populations are excited