The aim of our study was to employ fractal analysis for evaluation of ultrastructural changes during early stages of apoptosis. Apoptosis was induced in U251 human glioma cell line by exposure to UVB light. The cells were visualized by optical phase-contrast microscopy and photographed before the UV treatment, immediately after the treatment, as well as at 30 min intervals during 5h observation period. For each of the 32 cells analyzed, cellular and nuclear fractal dimension, as well as nuclear lacunarity, were determined at each time point. Our data demonstrate that cellular ultrastructural complexity determined by fractal dimension and lacunarity significantly decreases after the UV irradiation, with the nuclear lacunarity being a particularly sensitive parameter in detecting early apoptosis. Importantly, fractal analysis was able to detect cellular apoptotic changes earlier than conventional flow cytometric analysis of phosphatidylserine exposure, DNA fragmentation and cell membrane permeabilization. These results indicate that fractal analysis might be a powerful and affordable method for non-invasive early identification of apoptosis in cell cultures.
