Three types of evanescent wave-excited fluorescence microscopy. (a) Through-the-objective TIRFM, wherein a high numerical aperture (NA) objective lens is used to simultaneously generate the evanescent field at the cell-glass interface and view the cell. A laser light is directed and focused on the back focal plane, which then creates a refracted parallel beam approaching the interface in the small gap between objective and glass coverslip. TIR is achieved when the angle is greater than the critical angle (θ > θc); (b) Prism-based surface plasmon-excited TIRFM, wherein an incident light is directed onto a gold film via a prism, creating an electromagnetic field penetrating into the cell under resonance condition. The reflected beam is detected via a photodetector or imager for SPR measurement, while the excited fluorescence is collected using a separate objective; (c) Resonant waveguide grating-based TIRFM, wherein a grating is used to couple light into the waveguide, creating an evanescent field resulting from total internal reflection of the light beam. The excited fluorescence is collected using a CCD camera.
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