Visualizing photoactive molecules at electron miscroscopy.

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PS). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of new oxidizing chemical species (oxigen in the form of singlet, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase penetration through the plasma membrane, a PS may be modified by addition of chemical groups (e.g., acetate or phosphate): this causes quenching of the fluorescence emission and of the photosensitizing properties of the native PS. These modified compounds behave as fluorogenic substrates (FS) as inside the cell the bound residues are enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. Among the PS which may be modified to FS, two very efficient singlet oxygen producers, Rose Bengal (RB) and Hypocrellin B (HypB) have been especially investigated by our group. With the aim to detect photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with either RB-Ac or HypB-Ac, and took advantage of the fluorescence properties of the intracellularly restored PS to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and that they are converted into the native PS already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescìcular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-AC- or HypB-Ac-loaded cells. The halflife of oxidizing chemical species such as singlet oxygen is very short (from 1 ns to 1 ms) their mobility being limited to 1 to 30 nm: this implies that DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore DAB photoconversion promise to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.