The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.
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A. Rück, J. Breymayer, L. Lilge, A. Mandel, P. Schäfer, B. von Einem, C. von Arnim, S. Kalinina, "Two-photon luminescence lifetime imaging microscopy (LIM) to follow up cell metabolism and oxygen consumption during theranostic applications," Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104980A (23 February 2018);