Hyper-Rayleigh and hyper-Raman scattering background of liquid water in two-photon excited fluorescence detection

The detection of two-photon excited fluorescence is practically free from background caused by linear scattering because two-photon excited fluorescence occurs at a much shorter wavelength region than the excitation light. This property can be used to achieve ultrasensitive fluorescence detection. However, similar to linear scattering in the detection of one-photon excited fluorescence, the question arises whether background caused by nonlinear scattering may limit the detection sensitivity of two-photon excited fluorescence, In this work, quantitative comparisons between two-photon induced scattering of liquid water and two-photon excited dye fluorescence in a standard epifluorescence geometry show that the relative scattering background is typically reduced by orders of magnitude in two-photon excitation as compared to single-photon excitation with confocal detection, Hyper-Rayleigh and hyper-Raman (3400 cm(-1)) cross sections of liquid water have been measured to be 8 x 10(-62) and 7 x 10(-63) cm(4) . s/photon, respectively, at 840 Mn incident wavelength, with absolute values calibrated with respect to the known two-photon fluorescence excitation cross section of fluorescein.