Confocal spectral imaging analysis in studies of the spatial distribution of antitumour drugs within living cancer cells

Advanced instrumentation allowing the recording of confocal microfluorescence and micro-Raman spectra, with spatial resolution on the sub-micrometre scale, has been developed. Computer-controlled automatic scanning stage and a system of scanning the laser beam through the stage both permit the recording of well resolved spectral images of samples. Conventional images of species could be recorded with a CCD camera through the same optics. The developed software includes the possibilities of converting a spectra image into a conventional image and of assigning point-by-point the spectral data to the conventional image. This technique was applied to the analysis of the microfluorescent signals from living cancer cells treated wtih the antitumour drugs doxorubicin and mitoxantrone. It was possible to construct separately the spectral images corresponding to the distribution of drug-DNA complexes, the free drug and intrinsic signals from untreated cell. Consequently, quantitative comparative intracellular and even intranuclear localization of antitumour drugs was achieved. The differences in the intracellullar distribution of the drug-DNA complexes inside the cells are discussed in terms of biological efects.