Abstract: The bio-sample (e.g., a live cell) is labeled with a proper number of nanoparticles. Each nanoparticle is pre-co-doped with a controlled ratio of fluorophore donors and acceptors. Two laser pulses are applied to the bio-sample. The first laser pulse has a center wavelength near the peak of absorption spectrum of acceptors. The intensity of first laser pulse is adjusted such that FRET saturation occurs near the center of the focal spot. The focal spot of the first laser pulse is a diffraction-limited Airy disk that has the highest laser intensity in the center of the focal spot. The second laser has a center wavelength in the emission spectrum of acceptors and with a uniform intensity distribution throughout the focal spot. The fluorescence emission from acceptors after two laser pulses is from an area that is smaller than the diffraction-limited focal spot. Hence, a higher than diffraction-limit resolution is achieved.

Abstract: The bio-sample (e.g., a live cell) is labeled with a proper number of nanoparticles. Each nanoparticle is pre-co-doped with a controlled ratio of fluorophore donors and acceptors. Two laser pulses are applied to the bio-sample. The first laser pulse has a center wavelength near the peak of absorption spectrum of acceptors. The intensity of first laser pulse is adjusted such that FRET saturation occurs near the center of the focal spot. The focal spot of the first laser pulse is a diffraction-limited Airy disk that has the highest laser intensity in the center of the focal spot. The second laser has a center wavelength in the emission spectrum of acceptors and with a uniform intensity distribution throughout the focal spot. The fluorescence emission from acceptors after two laser pulses is from an area that is smaller than the diffraction-limited focal spot. Hence, a higher than diffraction-limit resolution is achieved.