SUBCELLULAR-LOCALIZATION OF SULFONATED TETRAPHENYL PORPHINES IN COLON-CARCINOMA CELLS BY SPECTRALLY RESOLVED IMAGING

Subcellular localization of the dye, 5,10,15,20-tetra(4-sulfonatopheny l)porphine (TPPS4) and the more hydrophobic dye, 5,10,15,20-tetra(1-su lfonatophenyl)porphine (TPPS1), in murine colon carcinoma cells was st udied by spectrally resolved imaging (SRI) combined with image process ing techniques, Spectrally resolved imaging enabled the acquisition of multipixel fluorescence spectra (>10(4)) from a single cell, Demarcat ion of specific localization sites and segregation of the irrelevant f luorescence were based on the pixel spectra and by operating the funct ions of spectral similarity mapping (SSM), principal component analysi s (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 th e photochemical effect, The dye TPPS4 was localized initially in lysos omal vesicles, and upon irradiation fluorescence was seen in the nucle us as well as in vesicles, Some of the vesicles were closely related t o the nucleus, as resolved by SSM, PCA and spectral classification, Ad ditional light exposure stimulated relocalization of TPPS4 into the nu cleus as well as into the nucleolus, which was clearly depicted by SSM and PCA, Spectral classification showed a third, weak residual cytopl asmic array around the nucleus, The dye TPPS1 concentrated in a Golgi- like complex and was resolved in the nuclear envelope and in small ves icles: it was not redistributed into other compartments upon photosens itization, Serum supplementation to the incubation media of colon carc inoma cells treated with TPPS4 or TPPS1 did not change the localizatio n 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 thei r interaction with serum proteins, was the main determinant of their b inding to the lysosomes, nucleus, nucleolus, nuclear envelope or Golgi .