Two-photon absorption cross-sections and time-resolved fluorescence imaging using porphyrin photosensitisers

Three porphyrin systems have been characterised for use in two-photon fluorescence imaging of biological samples. We have determined the two-photon absorption cross sections (σ₂) of the di-cation, free-base and metallated forms of hematoporphyrin derivative (HpD), hematoporphyrin IX (Hp9) and a boronated protoporphyrin (BOPP) using the open-aperture Z-scan and the two-photon induced fluorescence (TPIF) techniques at an excitation wavelength of 800 nm. The insertion of either protons or a metal ion into the macrocycle is shown not to significantly influence the σ₂ of the porphyrins. Two-photon time-resolved fluorescence images of C6 glioma cells transfected with a free-base form of the BOPP have been obtained as a function of the porphyrin concentration. These studies reveal a maximum useful porphyrin concentration for fluorescence imaging purposes of approximately 30 µg mL⁻¹.