Blank-Free Two-Photon Excited Fluorescence Detection

Trace-level impurities, such as those found in organic solvents and aqueous buffer solutions, are known to have little contribution to the two-photon excited fluorescence blank. Previously, the major source of interference in 90° fluorescence detection has been Rayleigh scatter at the laser wavelength. Various instrumental approaches have been used to minimize the amount of scattered radiation reaching the detector. Because two-photon excited fluorescence occurs primarily at the focal point of a lens, spatial isolation with microscope objectives has been used to maximize the signal-to-scatter ratio. Modulation of the excitation beam followed by second harmonic detection of the fluorescence also was shown to reduce scatter on the basis of frequency. Photon burst spectroscopy has recently been used to differentiate between fluorescence from single molecules and scatter background, while subnanosecond excitation of fluorophores has been combined with time-filtered detection to discriminate against instantaneous scatter. The reduction in background interference from these techniques has resulted in detection limits in the picomolar range.