Figure 1. Schematic optical diagrams of through-the-lens TIRF microscopy.

Excitation beams are shown in blue. Numbers indicate refractive indices of different materials found in the setup. (a) TIRF microscopy of adherent cells () on a cover glass (). Excitation beam on the right comes at the critical angle, . Excitation beam on the left has an angle of incidence, , greater than . (b) TIRF microscopy of adherent cells on a thin layer of silicone gel () on a cover glass. Excitation beam comes at an angle , which is the maximal value achievable with an NA = 1.46 TIRF objective, is refracted at the glass-gel boundary to an angle of , and is totally reflected at the gel-cell boundary, producing an evanescent wave with a penetration depth, nm for nm.