Characterization of p-bis(O-methylstyryl) benzene as a lifetime and anisotropy decay standard for two-photon induced fluorescence

We describe the fluorescence spectral properties of p-bis(O-methylstyryl)benzene (bis-MSB) as a standard for time-resolved measurements of two-photon induced fluorescence. Bis-MSB displays the same single exponential intensity decay in several solvents for one- and two-photon excitation. The anisotropy decay displays the same single correlation time of one- and two-photon excitation. The amplitudes of the anisotropy decay are distinct for one- and two-photon excitation. At some excitation wavelengths the anisotropy amplitude appears to be solely the result of one- and two-photon photoselection, but at shorter wavelengths the anisotropy amplitudes are not related by single constant factor. The absorption range of bis-MSB makes it a suitable standard for one- and two-photon excitation of intrinsic protein fluorescence and extrinsic fluorophores.