The role of triplet state relaxation in 1- and 2-photon excitation fluorescence microscopy

A significant limitation of fluorescence microscopy is the photodestruction of the fluorescent species, limiting the observation time and the total amount of detected fluorescence. This study presents a universally valid improvement of the fluorescence signal and at the same time a significant reduction of bleaching, exemplarily shown for the fluorescent label Atto532 (Rhodamine dye) and the green fluorescent protein GFP. The basic idea is to increase the triplet state relaxation of the fluorescent species by reducing the repetition rate of a pulsed laser. The results have implications for all types of single and multi-photon excitation microscopy, as well as for subdiffraction resolution STED-microscopy.