Characterization and performance of the femtosecond fluorescence up-conversion microscope

Abstract

The characterization and performance of the femtosecond fluorescence up-conversion microscope is reported in this paper. This new fluorescence microscope is a combination of the frequency up-conversion technique and a confocal optical configuration, which simultaneously achieves femtosecond time and nanometer space resolution. The femtosecond time resolution was evaluated by measuring the rise up of time-resolved fluorescence from a dye molecule, and it was 520 fs and 460 fs with 100× (N.A.=1.3) and 40× (N.A.=0.75) objective lenses, respectively. The best transverse (XY) resolution was 0.34 μm with the 100× objective lens for 400 nm excitation. An axial (Z) resolution as high as 1.1 μm was obtained for 600 nm fluorescence detection with a 50 μm pinhole and a 100× objective lens. The axial resolution was remarkably improved compared with ordinary confocal microscopes owing to the up-conversion process, which requires spatial overlap between the tightly focused gate and the fluorescence beams. Femtosecond time-resolved fluorescence measurements were performed for micro-meter sized particles in liquids, fluorescent beads and C519/toluene micro droplets, by using the laser trapping technique. The high potential of the fluorescence up-conversion microscope was demonstrated.