Subcellular localization of the
dye, 5,10,15,20-tetra(4-sulfonatophenyl)porphine (
TPPS4) and the more hydrophobic
dye, 5,10,15,20-tetra(1-sulfonatophenyl)porphine (TPPS1), in murine colon
carcinoma cells was studied by spectrally resolved imaging (SRI) combined with image processing techniques. Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (> 10(4)) from a single cell. Demarcation of specific localization sites and segregation of the irrelevant fluorescence were based on the pixel spectra and by operating the functions of spectral similarity mapping (SSM), principal component analysis (PCA) and spectral classification. The SRI revealed the fine details of the photochemical process that clarify some aspects of subcellular damage. The SRI depicted the differences between
TPPS4 and TPPS1 with respect to their initial localization and their fate at the end of the photochemical effect. The
dye TPPS4 was localized initially in lysosomal vesicles, and upon irradiation fluorescence was seen in the nucleus as well as in vesicles. Some of the vesicles were closely related to the nucleus, as resolved by SSM, PCA and spectral classification. Additional light exposure stimulated relocalization of
TPPS4 into the nucleus as well as into the nucleolus, which was clearly depicted by SSM and PCA. Spectral classification showed a third, weak residual cytoplasmic array around the nucleus. The
dye TPPS1 concentrated in a Golgi-like complex and was resolved in the nuclear envelope and in small vesicles: it was not redistributed into other compartments upon
photosensitization. Serum supplementation to the incubation media of colon
carcinoma cells treated with
TPPS4 or TPPS1 did not change the localization patterns. Pixel spectra of the two
dyes in the cells showed spectral shifts and expanded shoulders due to microenvironmental effects. Thus, the chemical nature of the sulfonated phenyl porphines, and not their interaction with
serum proteins, was the main determinant of their binding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi.