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Previously, we reported on the uptake and interaction of cytotoxic doxorubicin in MCF-7 breast cancer cells grown as standard 2-dimensional cell cultures. Now improved experimental techniques – including axial tomography and Light Sheet Fluorescence Microscopy (LSFM) – permit observation of single cells from any side as well as detection of individual layers in multi-cellular spheroids. Therefore, uptake of doxorubicin in the cell nucleus as well re-localization in the cytoplasm at longer incubation times is well documented. Based on a calcein-AM test, we could prove high cytotoxicity in 3D cell cultures at 48-96h after incubation. Simultaneously, disintegration of cell spheroids and formation of a degradation product became obvious. Fluorescence lifetime imaging microscopy (FLIM) is presently used to distinguish the fluorescence of doxorubicin and its degradation product, and Structured Illumination Microscopy (SIM) is suggested to improve resolution down to about 100 nm.