NONLINEAR DYNAMICS OF INTRACELLULAR METHYLENE-BLUE DURING LIGHT ACTIVATION OF CELL-CULTURES

Methylene blue (MB+) is a well-known dye in medicine and has been disc ussed as an easily applicable drug for topical treatment in photodynam ic therapy (PDT), Methylene blue can potentially be used as a redox in dicator to detect the important redox reactions that are induced durin g PDT. The kinetics of this process was analyzed on a subcellular leve l with confocal laser scanning microscopy, BKEz-7 endothelial cells we re incubated 4 h with 1 mu M MB+., The fluorescence dynamics of MB+ du ring irradiation with 633 nm light was observed with subcellular resol ution, Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s), Fluorescence was observed in the red channel of the laser scannin g microscope, Synchronously, the phase-contrast image was visualized w ith the green channel, Morphological changes could therefore be correl ated with the dynamics of MB+. In addition, the light-dose-dependent p hototoxicity at 633 nm irradiation was determined by viable cell count ing, After an induction period (phase I), fast fluorescent spikes coul d be observed in the whole cytoplasm, which decayed with a time consta nt of about 20 s (phase II), followed by a period of nearly constant f luorescence intensity (phase III) and exponential photobleaching (phas e IV), Phase II exhibits highly nonlinear kinetics, which is hypothesi zed to correlate probably with a nonlinear quantal production of react ive oxygen species (ROS), Morphological cell changes were not observed during phase II, During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light d ose applied within phase II was only sublethal in correlation with mor phological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.