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 present an analysis of the advantages and disadvantages of recording and monitoring of nonlinear strain waves in a solid waveguide made of a nonlinearly elastic material (polystyrene) using three techniques: off-axis digital holography with observation ‘in-transmission’ and ‘in-reflection’ and acoustical diagnostics utilizing piezoelectric transducers. Holographic ‘in-transmission’ recording was shown to provide more reliable determination of wave parameters owing to both higher resolution and sensitivity to longitudinal waves only. When monitoring the wave process by piezoelectric sensors attached to the lateral surface of the waveguide, the sensor sensitivity to shear waves did not allow us to isolate the contribution of longitudinal waves in a prolonged input part of the waveguide.
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 present detection of strain solitary waves in polystyrene waveguides using pump-probe digital holography. Analysis of spectral components of the detected longitudinal strain waves was performed at several locations of the waves inside the waveguide in the course of their propagation. The data obtained allowed us to evaluate attenuation of relatively short- and long-wavelength components and to demonstrate much faster decay of high-frequency wavelets in comparison with the long solitary wave. Along with the spectral analysis monitoring of variations of solitary wave shape in the course of its propagation was performed. The dependence of spectral composition of the strain wave on the energy of the initial shock wave showed an increase in the relative contribution of long-wave components with the strain wave energy.
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.
Accounting for nonlinear elasticity of modern materials becomes very important due to their rising operation at high dynamic loads. Generation of strain solitary waves (solitons for brevity) is one of the processes of interest, however details of a transformation of an initial impact into the soliton are not completely clear yet. In this paper we demonstrate the advantages of a combination of classical and digital holographic recording for investigation of the early stages of soliton formation. While classical realization of holographic interferometry allowed for visualizing sharp phase gradients representing, in particular, shock waves, digital recording supplied quantitative data on parameters of smoother disturbances evolving in the course of soliton formation. The applied holographic techniques allowed us to monitor the entire process of soliton formation, to visualize intermediate wave patterns and to obtain quantitative data on the resulting soliton.
Photodynamic inactivation (PDI) is known to be effective for treatment of various viral and bacterial infections. In view of the current COVID-19 pandemic the search for therapeutic modalities efficient against this particular virus is of high demand. PDI with photosensitizer solution applied in the oral cavity and throat by flushing and gargling was already demonstrated to be promising for reduction of viral load at early stages of COVID- 19 infection. In this report we present experimental results on detection of singlet oxygen generated using Radachlorin photosensitizer in nebulizer aerosol jet and on different biological surfaces modeling, in particular, mucous membranes of the respiratory tract. The lifetimes of singlet oxygen and photosensitizer triplet state were shown to depend noticeably on the surface type. Moreover the surface type was found to be strongly affecting the photosensitizer photobleaching kinetics, with mucous samples providing much slower bleaching.
We studied polarized fluorescence in biological coenzyme NADH in water-methanol solutions upon two-photon excitation with femtosecond laser pulses at 720 nm. The polarized fluorescence decay was recorded by a time correlated single photon counting (TCSPC) system. Fluorescence decay times, rotational diffusion time, fluorescence anisotropy, and the ratio of two pre-exponential factors have been determined and studied as a function of methanol concentration. The results obtained were interpreted on the basis of a model of NADH denaturation processes in solutions and can be used for modeling of NADH binding with various dehydrogenases in living cells.
We present a comparative analysis of photodynamic-treatment induced changes in optical parameters of cancer cells obtained from individual patients with three solid tumor localizations. Accumulation of photosensitizer inside living cells was validated using far-field fluorescence microscopy. Measurements of their optical characteristics were performed by means of digital holographic microscopy. The quantitative analysis of cell death dynamics performed by digital holographic microscopy was shown to be promising for investigation of cells resistivity to treatment. It was shown that both the photosensitizer accumulation and post-treatment dynamics of average phase shift may differ significantly in cell cultures obtained from different tumor localizations and different patients. Some of the cell cultures demonstrated very low or even no response to treatment.
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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