Two-photon laser microscopy

A laser scanning microscope, comprising

• stage means for receiving a target material having an absorption energy level peak responsive to single photon excitation by light of a predetermined wavelength;
• lens means positioned to direct light to said stage means and having an object plane in target material at said stage means;
• a laser source of subpicosecond a laser light pulses, said laser light having a wavelength about twice said predetermined wavelength;
• mirror means directing said laser light pulses along an optical path including said lens means to cause said pulses to impinge on target material at said object plane, said lens means focusing said laser light pulses on a focal point in the target material, the intensity of said pulses producing in the region of said focal point a two-photon excitation energy level equivalent to the single-photon excitation energy level which corresponds to said single photon absorption peak.

A method of fluorescence microscopy by a two-photon excitation technique, comprising:

• providing a sample containing fluorescent molecules which radiate photons of a first characteristic energy;
• illuminating said sample with a beam of rapidly repeating, intense, subpicosecond pulses of laser light comprising photons of a second characteristic energy, wherein said second characteristic energy is about one-half said first characteristic energy;
• focusing said illumination to a focal point having a submicron diameter with said sample to produce an illumination intensity sufficiently high at said focal point to produce molecular excitation and fluorescence of said sample by simultaneous absorption of two incident photons;
• scanning the submicron diameter focal point of said beam in a raster pattern through said sample;
• and
• detecting the fluorescence produced by said sample.