Patent ID: 7863585

Claim:
A method of high spatial resolution imaging a structure of interest in a sample, the method comprising the steps of: marking the structure of interest with molecules of a fluorescent dye; selecting a first wavelength for excitation light which excites the molecules of the fluorescent dye via a multi photon process for spontaneous emission of fluorescent light; focussing pulses of the excitation light of the first wavelength into the sample to excite those molecules of the fluorescent dye which are present in at least one focal area of the focussed excitation light for the spontaneous emission of fluorescent light; selecting a second wavelength, which is shorter than the first wavelength, for de-excitation light which de-excites excited molecules of the fluorescent dye prior to their spontaneous emission of fluorescent light; during a plurality of the pulses of the excitation light, continuously directing a pattern of the de-excitation light of the second wavelength onto the sample to de-excite excited molecules of the fluorescent dye, which are located outside at least one measurement area which is a fraction of the at least one focal area; and recording the fluorescent light spontaneously emitted by the molecules of the fluorescent dye in the sample.