New long wave and highly efficient two-photon fluorophores for multiphoton microscopy

An important ingredient in improving Multi Photon Laser Scanning Microscopy, MPLSM, is the development of efficient exogenous two-photon fluorescent (TPF) probes. Here we report on a new class of two-photon fluorophores, specifically designed in order to maximize their efficiency in potential MPLSM applications. The fluorophores possess a symmetric Donor-Acceptor-Donor (D-pi -A-pi -D) structure with varying conjugation length and have strong donors and accepters. We have studied the two-photon excitation (TPE) properties of these fluorophores and found the following properties: a) Very large two-photon absorption coefficients (delta > 1000GM); b) Peak TP excitation wavelength which are strongly shifted to the red (lambda similar to 1 mum); c) Large fluorescence quantum efficiency; d) Large Stokes shifts of the fluorescence bands. These properties make them particularly suitable for imaging thicker samples, relying on the large improvement in TPE cross-sections and the reduced attenuation at both the excitation and emission wavelengths. We also describe TPE fluorescence anisotropy experiments revealing the tensorial shape of the fluorophores.