Patent ID: 7298476

Claim:
A method for exceeding diffraction-limit resolution in laser scanning microscopy using fluorescence-resonance-energy-transfer-induced-emission-depletion (FRET-IED), said method comprising: preparing a sample by labeling the sample with a plurality of nanoparticles, each of the nanoparticles being co-doped with a controlled ratio of donors and acceptors; placing the sample under an objective lens to be laser scanned, wherein the objective lens having an adjustable numerical aperture; applying a first laser pulse to form a focal spot of the first laser pulse on a location of interest of the sample with a desired intensity, wherein the focal spot of the first laser pulse is formed by setting the adjustable numerical aperture to highest value in accordance with highest resolution based upon the diffraction-limit; applying a second laser pulse to the focal spot of the first laser pulse after a pre-determined time interval; and detecting and recording fluorescence emission from the acceptors located within a central small circular area inside the focal spot of the first laser pulse.