Method of microscopic observation

A method of microscopic observation, comprising:

• dying a specimen with molecules each having at least three quantum states inclusive of a ground state;
• and
• observing the specimen through a microscope, said microscope comprising:
• a light source device constructed and arranged to emit light of a wavelength λ1 that excites the molecules from a ground state to a first-level excited state and light of a wavelength λ2 that excites the molecules from the first-level excited sate to a second-level excited state, said second-level excited state being higher in energy than said first-level,
• a focusing optical system for focusing the light of the wavelength λ1 and the light of the wavelength λ2 on the specimen,
• a photodetector for detecting luminescence which occurs according as the molecules, with which the specimen is dyed, decay to return to the ground state;
• and
• an irradiation overlapping assembly constructed and arranged to overlap a region irradiated with the light of the wavelength λ1 with a region irradiated with the light of the wavelength λ2 so that a region in which the luminescence accompanying a decay of the molecules from the first-level excited state to the ground state is limited by said irradiation overlapping assembly through which the specimen is irradiated with the light of the wavelength λ1 and the light of the wavelength λ2,
• wherein the molecules with which the specimen is dyed are used as fluorescence probe molecules and have such a property that a relaxation process with heat emission is predominant over a relaxation process with fluorescence emission in a transition of decay from a higher-level energy state other than the first-level excited state to the ground state, and
• wherein the wavelength λ2 is different from a wavelength of the luminescence emitted from the specimen.