TIRF - Total Internal Reflection Fluorescence

Introduction to the basics of TIRF

The optical phenomenon of total internal reflection (TIR) can be observed in everyday life. The century old handcraft of gemstone cutting is a typical example. Keeping the light within a diamond and creating as many reflections as possible before it exits, produces the so called "fire" of a gemstone. The light undergoes multiple internal reflections before it gets refracted. The same principle is used in modern fiber optic data translation.

The goal of using total internal reflection fluorescence (TIRF) spectroscopy in biological applications is to study events close to the interface of two different media. A simple example would be a droplet of water on a glass plate. There is no refraction if you follow a beam along the optical axis (α1=0°) of such a system. Increasing the angle of the incoming beam (β2=30°) would lead to refraction away from the optical axis. The reason for this is that the refractive index of glass is bigger than the one of water. If you further increase the angle of the incoming beam above the so called "critical angle", total reflection occurs (γ3> α_critical). The critical angle is defined by the density (the refractive index η) of the two media forming the interface. The relationship between the angle of the incoming and the refracted beam is given by the law of refraction:

Assuming that the beam travels from the optically denser medium into the less dense medium, the critical angle is calculated as follows:

For the interface of our example formed by water and glass, the refractive indices are η water = 1,333 and η quartz = 1,544, respectively. Hence the critical angle for this system is 59,7°.