Reduction of higher-order photobleaching in two-photon excitation microscopy

A theoretical microscopic technique is proposed that may reduce multiphoton interaction in the excitation volume of a two-photon microscope. Since higher-order photobleaching is common in two-photon excitation microscopy, the study of thin samples is limited by increased photobleaching and photodamage. This limitation is elevated by using even coherent state light. The advantage of even coherent state light is that only excitation due to an even number of photons can survive. The very first nonzero even excitation (two-photon) can be isolated from the nearby one- and three-photon excitation. Hence the photobleaching due to one- and three-photon excitation can be eliminated and higher-order processes can be minimized owing to their small molecular cross section.