Two-photon fluorescence cross-section measurements calibrated using hyper-Rayleigh scattering

Summary form only given. There has been renewed interest in two-photon fluorescence (2PF) as a result of its recent application in 2PF microscopy, as well as other applications. Despite the long history of 2PF studies, absolute determinations of 2PF cross sections remain infrequent and difficult, and substantial disagreements between published values of 2PF cross sections exist. We report a novel method for measuring the absolute 2PF cross section that eliminates key sources of potential errors in existing methods. Our method compares two different two-photon scattering processes in the same sample under identical conditions, 2PF and hyper-Rayleigh scattering (HRS). Although the dependence on experimental parameters of the intensity of 2PF and HRS is almost identical, the spectrum of the light produced by HRS is sharply peaked near twice the incident laser frequency, whereas the 2PF emission spectrum is broader and shifted to lower frequencies than the HRS spectrum. The one critical aspect is that an accurate value for the hyperpolarizability /spl beta/ must be available. Recent work has provided accurate HRS /spl beta/ values for a number of chromophores in solution. The only measurement that is needed to determine the 2PF cross section calibrated in terms of the HRS cross section is the relative integrated intensity of the 2PF and HRS components of the scattered light spectrum.