TIRF Microscopy

Total internal reflection fluorescence microscopy (TIRFM) is a highly sensitive technique to perform functional investigations in living cells. The high signal to noise ratio and a resolution in z direction of usually 70-250 nm above the coverslip/water interface allows to visualize and to analyse vesicles transport and signalling events, as well as kinetic studies and single molecules detection. Physical background is the total reflection of a laser beam at the interface of glass and water and the resulting electromagnetic wave, the so-called evanescent field. The energy of the evanescent field decreases exponentially with the distance of the interface coverslip/water and allows exciting fluorochromes.