Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocal fluorescence microscopy

Photodynamic therapy (PDT) is a novel cancer therapy that uses light-activa ted drugs (photosensitizers) to destroy tumor tissue. Reactive oxygen speci es produced during PDT are thought to cause the destruction of tumor tissue . However, the precise mechanism of PDT is not completely understood. To pr ovide insight into the in vitro mechanisms of PDT, we studied the subcellul ar localization of the photosensitizer HOSiPcOSi(CH3)(2)-(CH2)(3)N(CH3)(2) (Pc 4) in mouse lymphoma (LY-R) cells using double-label confocal fluoresce nce microscopy, This technique allowed us to observe the relative distribut ions of Pc 4 and an organelle-specific dye within the same cell via two, sp ectrally distinct, fluorescence images. To quantify the localization of Pc 4 within different organelles, linear correlation coefficients from the flu orescence data of Pc 4 and the organelle-specific dyes were calculated. Usi ng this measurement, the subcellular spatial distributions of Pc 4 could be successfully monitored over an 18 h period. At early times (0-1 h) after i ntroduction of Pc 4 to LY-R cells, the dye was found in the mitochondria, l ysosomes and Golgi apparatus, as well as other cytoplasmic, membranes, but not in the plasma membrane or the nucleus. Over the next 2 h, there was som e loss of Pc 4 from the lysosomes as shown by the correlation coefficients, After an additional incubation period of 2 h Pc 4 slowly increased its acc umulation in the lysosomes. The highest correlation coefficient (0.65) was for Pc 4 and BODIPY-FL C-5 ceramide, which targets the Golgi apparatus, and also binds to other cytoplasmic membranes. The correlation coefficient was also high (0.60) for Pc 4 and a mitochondria-targeting dye (Mitotracker Gr een FM). Both of these correlation coefficients were higher than that for P c 4 with the lysosome-targeting dye (Lysotracker Green DND-26). The results suggest that Pc 4 binds preferentially and strongly to mitochondria and Go lgi complexes.