The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X-100 micelle solution have been examined using near-infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.

The spectroscopy and photochemistry of protoporphyrin IX in ethanol and in Triton X‐100 micelle solution have been examined using near‐infrared two‐photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. We have determined that the photochemistry subsequent to TPE is very similar to that found for one‐photon excitation. Moreover, the photoproducts observed possess very intense TPE fluorescence spectra, which allows their detection at low relative concentrations.

Two-photon excitation photodynamic therapy (TPE-PDT) is being developed as an improved treatment for retinal diseases. TPE-PDT has advantages over one-photon PDT, including lower collateral damage to healthy tissue and more precise delivery of PDT. As with one-photon PDT, there can be local photochemical depletion of oxygen during TPE-PDT. Here, we investigate model systems and live cells to measure local photosensitizer photobleaching and through it, infer local oxygen consumption in therapeutic volumes of the order 1 microm3. Multilamellar vesicles (MLV) and African green monkey kidney (CV-1) cells were used to study the TPE photobleaching dynamics of the photosensitizer, Verteporfin. It was found that in an oxygen-rich environment, photobleaching kinetics could not be modeled using a mono-exponential function, whereas in hypoxic conditions a mono-exponential decay was adequate to represent photobleaching. A biexponential was found to adequately model the oxygen-rich conditions and it is hypothesized that the fast part of the decay is oxygen-dependent, whereas the slower rate constant is largely oxygen-independent. Photobleaching recovery studies in the CV-1 cells support this hypothesis.

Fluorescence correlation spectroscopy (FCS) is a highly sensitive and selective technique with which one can analyze highly localized environments using the fluctuations in the average fluorescence of a probe fluorophore. Two-photon FCS has been proposed as a method to minimize photodamage to the probes and living organisms under observation. While multiphoton excitation reduces photodamage outside the focal volume, photodynamic effects within the volume can be substantial. The dynamics occurring in the focal excitation volume of a two-photon confocal microscope are examined using FCS of dye-loaded, 100 nm diameter polystyrene spheres. Multiphoton photodynamic events are observed which cannot be explained either by simple local solution heating or by intersystem crossing within a single excited dye molecule. The laser power dependence of photodynamics process suggests that a four-photon absorption by polystyrene and subsequent ablation of the sphere is the mechanism of fluorescence-reducing photodamage.

The spectroscopy and photochemistry ofprotoporphyrin IX (PpIX) in ethanol and in Triton X-100 micelle solution and Verteporfin in methanol and Triton X-100 micelle solution have been examined using near infrared two-photon excitation (TPE). TPE will allow photodynamic therapy with highly localized light dosage. For PpIX, we have determined that the photochemistry subsequent to TPE is very similar to that found for one-photon excitation. Moreover, the photoproducts observed possess very intense two-photon excitation fluorescence spectra, which allows their detection at low relative concentrations. Verteporfin displays photodynamic behavior in methanol similar to that of PpIX in ethanol. However, in micelle solution Verteporfin exhibits photodynamic behavior indicative oftwo sensitizer populations, excimers and monomers. Photochemical models are presented.