MPE IMAGING OF THE PRIMARY EVENTS OF Photo Dynamic Therapy (PDT)

Photodynamic therapy (PDT) is a novel cancer therapy that selectively kills tumor cells by producing cytotoxic oxygen species via excitation of a photosensitive drug. One such drug, Photofrin has been approved in the United States for the treatment of esophageal cancer. We have demonstrated two-photon excitation of Photofrin in cellular monolayers of EMT6/Ro and HeLa cells. In the left figure of the next section, fluorescence emission in the wavelength range of 580-680 nm is shown from sensitized EMT6/Ro cells excited with 850 nm radiation. The cells were incubated for 40 minutes at 37C in media containing 5 µg/ml of Photofrin. The average excitation power was approximately 10 mW at the sample. While a great deal of research on the fundamental photophysical events of PDT has been conducted at the cellular level, less quantitative information is available in intact tumor systems. This information is critical for the proper definition and administration of photodynamic dose in vivo. To observe such relevant factors as the photosensitizer distribution, photosensitizer bleaching during therapy, and the effects of PDT on a variety of cellular processes in a realistic tumor model, we obtained three-dimensional fluorescence images of multicell tumor spheroids with multiphoton microscopy. The spheroid is an a vascular spherical aggregate of tumor cells grown in suspension culture to diameters ranging from hundreds of microns to millimeters. This distance is typical of the intercapillarys pacing encountered in tumors in vivo and, consequently, diffusion distances for cellular metabolites such as oxygen and glucose are preserved in this model.

While the size of the spheroid is optimal for a model tumor system, autofluorescence imaging of a radial section with conventional one-photon microscopy would lead to prohibitively large levels of UV exposure. The right figure (B) demonstrates the optical sectioning capability of multiphoton microscopy. Autofluorescence (350 –580 nm) resulting from 720 nm excitation focused 40 µm into a 400 µm diameter spheroid is shown (20X 0.75NA objective lens). In this particular image, a central region of necrosis (consistent with the experimental growth conditions) is seen to be surrounded by a viable rim several cell layers thick. Since photosensitizer fluorescence and autofluorescence occur in different wavelength intervals and both of these can be simultaneously excited with near infrared radiation, MPE microscopy should be ideally suited for observing the effects of PDT in intact tumor systems. Of particular interest is the distribution of ground-state molecular oxygen during therapy. Since reactive oxygen species are the source of the cytotoxic effects, PDT can quickly deplete a tumor of molecular oxygen during therapy if the rate of reaction exceeds the rate of diffusional resupply. The development of such a therapy induced anoxia has been linked with reduced therapeutic efficacy in vivo. Future experiments will seek to determine to what extent changes in cellular autofluorescence during therapy are correlated with changes in the local oxygen tension. This will be done by using Clark-style micro-oxygen electrodes (currently the gold standard in oxygen sensing) with tip diameters of approximately 5 µm to directly map the radial distribution of molecular oxygen in a multicell spheroid under a variety of controlled environmental conditions. Another closely related topic of research involves the quantitative determination of the sources of autofluorescence in neoplastic tissue. The fact that the autofluorescent signature of neoplastic tissue can differ appreciably from its normal counterpart offers a potentially noninvasive means of diagnosing a variety of abnormal conditions. While several empirical algorithms for performing such a diagnosis in vivo have been developed, the biochemical origins of these differences remain a mystery. Combining multiphoton microscopy with microfluorimetry (see Panel 6-C) allows the unique identification of autofluorescent species with inherent three-dimensional resolution. Because a wide variety of endogenous fluorophores can be excited and analyzed with this technique, a quantitative biochemical analysis of autofluorescence from diseased tissue should be possible.