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.
Accumulation of Radachlorin photosensitizer in 3T3 cells was evaluated using holographic tomography and fluorescence microscopy. Comparative analysis of images obtained by these techniques evidences drug accumulation in small intracellular structures located primarily in the juxtanuclear area.
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.
The paper presents results on the response of living HeLa cells in vitro to low-dose photodynamic treatment with Radachlorin photosensitizer. Quantitative monitoring of variations of optical and morphological parameters of cells was performed by means of digital holographic microscopy and assisted with observations in confocal fluorescent microscope. The statistical analysis of the results obtained demonstrated significant morphological changes of cells along with invariable dry mass. The AO/EB standard test validated cell membrane integrity and demonstrated cells rounding and membrane blebbing. These data allow us to assume apoptosis as a major pathway of cell death activated in our experimental conditions.
Monitoring of variations in morphological characteristics of cultured HeLa cells after photodynamic treatment with Radachlorin photosensitizer is performed by means of digital holographic microscopy. The observed dose-dependent post-treatment variations of phase shift evidence threshold effect of photodynamic treatment and allow for distinguishing between necrotic or apoptotic pathways of cell death. Results obtained by holographic microscopy were confirmed by means of far-field optical microscopy and confocal fluorescence microscopy with commonly used test assays.
Measurements of average phase shifts introduced by living HeLa cells to probe wave front were carried out. Variations of this value were monitored in the course of morphological changes caused by photodynamic treatment at various irradiation doses. Observations of changes in living cells were also performed by means of far field optical microscopy and confocal fluorescent microscopy. Quantitative analysis of the data obtained shows that average phase shift introduced by the cells may either increase or decrease depending upon major parameters of the treatment.
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