Total Internal Reflection Fluorescence Microscopy (TIRF)

In TIRF microscopy, an evanescent  light wave is used to illuminate the sample in close proximity to the coverslip. The background fluorescence contribution from above and below the focal plane is therefore removed, thus largely improving the axial resolution and the  signal to noise ratio of the imaging system. The evanescent wave is generated when light hits the interface between two media with different refractive indexes at the angle of total internal reflection, and typically only propagates up to 200nm into the sample. TIRF microscopy is used for imaging thin samples close to the coverslip, single molecule dynamics, diffusion of membrane bound molecules, etc.