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How Multiphoton Excitation can Illuminate Biophysics

Multiphoton molecular excitation by the strongly focused femtosecond pulses of infrared light generated as an 80 MHZ pulse train by a mode locked laser provides intrinsic submicron three dimensional spatial resolution of fluorescence excitation and photochemistry for laser scanning fluorescence microscopy. Because two-photon excitation requires simultaneous (∼10-16 seconds), absorption of two-photons focused laser intensities of about 1022 photons/cm2s are required. Since the rate of absorption is proportional to the square of the intensity, excitation is limited to the focal volume and is negligible elsewhere along the double cone of the focused illumination. Therefore, out of focus photodamage and fluorescence are generally negligible and laser scanning fluorescence microscopy with multiphoton excitation is intrinsically three dimensionally resolved with no out of focus background. Since the appropriate wave lengths are infrared for multiphoton excitation of ultraviolet or visible absorbing molecules, out of focus photodamage is eliminated. This allows imaging of useful ultraviolet absorbing indicators, vital DNA stains and autofluorescence in living cells with minimal, but not necessarily negligible, photodamage.