Monitoring of PpIX-induced death dynamics in HeLa cells by digital holographic tomography

We present the analysis of the response of human cervix epidermoid carcinoma HeLa cells to photodynamic treatment with protoporphyrin IX (PpIX) synthesized in living cells under accumulation of 5-aminolevulinic acid (5-ALA). PpIX synthesis was estimated by means of confocal uorescent microscopy basing on its uorescence intensity in the red wavelength range. The cell death dynamics and pathways caused by intracellular generation of reactive oxygen species under laser irradiation of photosensitized cells were examined using digital holographic tomography. These processes were studied both at early stages of cells death on living specimens and at later stages on paraformaldehyde-fixed specimens. Statistical analysis of changes in cellular morphology monitored using holographic tomography allowed us to conclude on cell death pathways, observed at different irradiation doses. Data obtained by holographic tomography have been validated by a standard AO/EB test for cell membrane integrity conducted using the confocal fluorescence microscope.