Fluorescence photoconversion of diaminobenzidine as a tool for detecting photosensitizing molecules at transmission electron microscopy

Photosensitizers (PSs) are chemical compounds able to absorb light and dissipate energy through photochemical processes producing highly unstable chemical species (i.e., singlet oxygen, free radicals or reactive oxygen species) that can damage the cell structures eventually inducing cell death. PSs may be modified by addition of suitable chemical groups (such as acetate or phosphate) to increase their intracellular accumulation: the photophysical and photochemical properties of the native PSs are affected, but the modified compounds behave as fluorogenic substrates (FSs) since once inside the cell the added groups are removed by cellular esterases and the native PS characteristics are restored. In the attempt to localize photoactive molecules at transmission electron microscopy, HeLa cells were loaded with two different FSs, Rose Bengal acetate or Hypocrellin B acetate, and the photophysical properties of the intracellularly restored PS molecules were exploited to photoconvert diaminobenzidine into an electrondense product. By this approach we demonstrated that these FSs enter the cells by endocytosis, being rapidly converted into the native PSs at the cell surface. PS molecules were also found in endosomes, lysosomes and multivescicular bodies, as well as free in the cytosol. This ultrastructural localization of the photoactive molecules accounts for the multiorganelle photodamage induced by irradiation of FS-loaded cells