The response of cells of different origin to photodynamic treatment in vitro was studied using two realizations of quantitative phase imaging (QPI): off-axis digital holographic microscopy and spatial light interference microscopy (SLIM), and using fluorescence lifetime imaging microscopy (FLIM). Holographic techniques were shown to allow noninvasive monitoring and analysis of the response of both individual cells in a sample and their entire population to photodynamic treatment. Dynamics of changes in the phase shift introduced by cells provided information on cell death type and rate. Utilization of a low-coherence radiation source in the SLIM realization ensured reduced measurement error due to an absence of coherent noise. Changes in the fluorescence intensity and decay time of the applied chlorin-based photosensitizer in cells were shown to be due to photobleaching of the photosensitizer, rather than to intracellular processes occurring in the course of cell death. The observed variations in optical and morphological parameters of cells as a function of treatment dose were shown to conform to the specific cell death pathways. The advantages and disadvantages of each technique are discussed.
We report investigation of HeLa cells’ response to photodynamic treatment in vitro using three realizations of quantitative phase microscopy. Reconstruction of phase images was performed by means of off-axis digital holographic microscopy with coherent HeNe laser source, and two approaches utilizing partially coherent illumination: transport of intensity equation (TIE) and spatial light interference microscopy (SLIM). All of these methods can be successfully used for analysis of optical and morphological characteristics of living cells and cells exposed to photodynamic treatment, however each technique has its own advantages and disadvantages in implementation and data processing.
We report experiments on photodynamic treatment of HeLa cells with 5-ALA. Intracellular localization of generated PpIX was determined and combinations of treatment parameters providing major pathways of cell death were obtained using 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.
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