TIRF, Total Internal Reflection Fluorescence microscopy is a method of exciting fluorophores very close to a surface (e.g., coverslip), without exciting molecules anywhere else in the sample. Using this method, researchers perform experiments designed to investigate molecular phenomena in or on membranes of cells that are adherent to the coverglass. Molecules further into the cell "see" no electromagnetic energy, and do not fluoresce.

The phenomenon is based on the concept of Snell's Law whereby light impinging on a surface can reach a certain angle (or greater) where it completely reflects off that surface. This Critical Angle of incidence is a function of the refractive index of the two media.

Snell's Law states:

Where i = incident medium or angle and t= transmitted medium or angle.

The Critical angle occurs when sin(theta-t) goes to 90 degrees. Under these conditions Snell's law becomes:

Snell's equation tells us only where the light will go.

To find out how much of the light (or energy) will go in all cases, either reflected or refracted, one needs to use equations described by Augustin-Jean Fresnel: the Fresnel Equations.

These equations describe light waves as Electric and Magnetic Vectors. What is important to understand is that the intensity of energy transmitted through an interface, when a light wave impinges on that interface is calculated using the Fresnel equations, and that this energy can be calculated knowing only:

• the Refractive Index of the materials
• the Angle of incidence

Implications of the Fresnel Equations