This invention relates to a method for the two-photon excitation of long-lived fluorescent or phosphorescent dyes with long low-power pulses. In the said method time-resolved detection is used to detect long-lived dyes. In the said method long-lived dyes are excited to the excited state with light pulses of long duration via double-photon absorption. In the two-photon absorption process the chromophor of the dye molecule is excited through the summation of the energies of two or more photons when they are simultaneously absorbed. As the said method is based on the use of pulses of long duration and on time-resolved detection the peak power of the pulses may be kept low. Long pulses of low power can be produced with a great variety of low-power light sources. The light source may in this case be e.g. a semiconductor laser. The excitation technique according to the invention may exploit the doubling or multiplication of the excitation wavelength or the nonlinearity of the two-photon absorption process. The method may be used for the detection of the fluorescence or phosphorescence from long-lived dyes for the determination of biospecific binding in applications of analytical or microscopic methods. <IMAGE>
