Super-resolution fluorescence microscopy by up-conversion-depletion using two color lasers

Summary form only given.We propose a novel scanning fluorescence microscope capable of breaking the diffraction resolution limit by up-conversion process with two-color pump and erase lasers. Most of dye molecules with an S/sub 1/ state generated by irradiating of a laser (i.e., pump laser,) decay to the ground state through fluorescence process. On the other hand, the radiative relaxation of the molecules with a higher excited S/sub 1/ state to the ground state is almost inhibited. By irradiating of pump beam and another laser (i.e., erase laser,) for S/sub n//spl larr/S/sub 1/ photo-excitation at the same time, the molecules with the S/sub 1/ state are up-converted, and the fluorescence can be reduced. If the focused pump and erase beams are partially overlapped on a sample stained by dye molecules, the fluorescence from the region irradiated by both lasers is inhibited. Only the innermost region of the maximum of pump laser contributes to the fluorescence signal. Consequently, a radius of fluorescence signal becomes smaller than the diffraction limit of the pump laser. By use of the up-conversion with two-photon-excitation concerning the two-color pump and erase lasers, a laser scanning microscope with a super-high resolution; i.e., super-resolution microscope could be expected.