Two-photon characterization and microscopy of porphyrin photosensitisers

The benefits of two-photon fluorescence microscopy of biological samples are vast arising from the utilisation of low energy light. The two-photon absorption cross sections (sigma(2)) of the di-cation free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) are obtained to ascertain their effectiveness as fluorophores for use in two-photon microscopy. The open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques, each capable of providing information regarding the nonlinear absorption, are employed to determine sigma(2) of the various porphyrins at an excitation wavelength of 800 nm. A significant disparity in the determined values of sigma(2) using the two methods is observed. This is largely attributed to the common requirement of higher concentrations used in the open aperture Z-scan method compared with TPIF techniques. Values of sigma(2) obtained from the Z-scan experiments are in the order of 10 GM, whilst those obtained from the TPIF experiments are in the order of 200 GM. Insertion of either protons or metal ions into the macrocycle does not enhance the sigma(2) of the porphyrins. Successful two-photon induced fluorescence imaging of BOPP free-base loaded G6 glioma cells is achieved, confirming the usefulness of this porphyrin in two-photon microscopy.