Through-the-objective TIRF microscopy: (A) schematic illustrating the formation of an evanescent wave by a laser beam directed through a high-NA objective to undergo total internal reflection at the interface between a glass coverslip and aqueous medium. Fluorescence emitted by a fluorophore excited by the evanescent wave is collected back through the same objective. (B) Layout of a TIRF imaging system based on an inverted microscope. Translation of the focusing lens is used to control the position of the excitation laser spot at the objective back aperture and thereby alter the angle of incidence at the interface between the coverslip and aqueous medium. The system includes a separate deep-red laser for feedback control of microscope focus.