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.
The influence of solution polarity and viscosity on fluorescence decay parameters in free and protein-bound NADH was studied. The fluorescence in NADH dissolved in water, methanol, ethanol, propylene glycol, and alcohol dehydrogenase-containing solution was two-photon excited by femtosecond laser pulses at 720 nm and recorded by means of the time-correlated single photon counting (TCSPC) method. Fluorescence decay times and corresponding weighting coefficients were determined by fit from polarization-insensitive fluorescence decay experimental signals. The fluorescence decay times τ1 and τ2, and weighting coefficients a1 and a2 were found to depend significantly on the solution type. A model describing the dependence of the fluorescence decay parameters on the microenvironment in solutions with different polarity and viscosity has been developed. According to the model, the heterogeneity in the measured fluorescence decay times in NADH and the time values were closely related with the charge distributions in the cis and trans configurations of the nicotinamide ring that result in different electrostatic field and different non-radiative decay rates. The influence of solution polarity and viscosity on the measured fluorescence decay times was investigated. As shown in high viscous solutions the increase of fluorescence decay times in NADH was mostly due to slowing down of the nuclei motions during the vibrational relaxation and intramolecular nuclear rearrangement whereas in low viscous solutions the fluorescence decay times follow the change of solution polarity.
The paper presents the study of anisotropic fluorescence decay of FAD in water-methanol solutions of different concentrations in the range of 0-80%. The fluorescence decay parameters: decay times, corresponding weighting coefficients, fluorescence anisotropy, and rotation diffusion time have been determined from experiment using the TCSPC method as function of methanol concentration and analyzed. The fluorescence kinetics demonstrated doubleexponential decay with two fluorescence decay times of about 2 and 4 ns. The decay times τ1 and τ2 were found to be the same under excitation at 450 nm and at 355 nm within experimental error bars and were practically independent of methanol concentration. The fluorescence anisotropy under 450 nm and 355 nm excitation was determined to be about 0.35 and 0.23, respectively indicating that the directions of the excitation transition dipole moments via the first and the second absorption bands differed significantly from each other. Also, the anisotropy was found to be practically independent of methanol concentration. The rotational diffusion time was proportional to the solution viscosity at lower methanol concentrations up to 40%, and reached a plateau at higher concentrations while the solution viscosity dropped down. This behavior was explained due to the increase of FAD solvation in the MeOH-water solution under the condition of FAD unfolding due to the denaturation effect.
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 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.
Fluorescence kinetics of the biological cofactor flavin adenine dinucleotide (FAD) in water-methanol solutions at 0%, 20%, 40%, 60% and 80% methanol concentration have been investigated. Fluorescence lifetimes, corresponding weighting coefficients, anisotropy, and rotational diffusion time were determined from experiment through analysis of the polarized fluorescence decay excited by picosecond laser pulses. The dependence of the fluorescence parameters on solution polarity and viscosity has been analyzed.
A novel polarization-modulation transient method has been developed for studying fast anisotropic relaxation in electronic excited states of polyatomic and biologically relevant molecules under excitation with femtosecond laser pulses. The method is based on the modulation of pump beam polarization with a photo-elastic modulator and detection of an anisotropic contribution to the transient signal by a highly-sensitive demodulation balanced scheme. The method was tested on aqueous solution of coenzyme NADH (nicotinamide-adenine-dinucleotide) pumped at 360 nm and probed at 720 nm. Anisotropic vibrational relaxation and rotational diffusion have been observed in the sub-picosecond time domain. The method significantly enhances the accuracy of transient measurements and allows for recording of high-quality signals at low energy (a nJ) pump pulses.
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.
The luminescence spectrum of aqueous solution of Radachlorin photosensitizer in the near IR spectral range (950-1350 nm) has been determined at the excitation in both the Soret and Q absorption bands. Major sources of the recorded luminescence were analyzed. Kinetics of photosensitizer and singlet oxygen phosphorescence signals were studied by means of time-resolved spectroscopy. The corresponding characteristic lifetimes were determined.
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.
The paper presents results on singlet oxygen detection in aqueous solutions of a photosensitizer based on the reconstruction of 3D temperature gradients resulting from nonradiative deactivation of excited oxygen molecules. 3D temperature distributions were reconstructed by means of the inverse Abel transformation from a single digital hologram in the case of cylindrically symmetric distribution of the temperature gradient and using holographic tomography algorithm with filtered back projection in the case of nonsymmetrical distribution. Major features of the applied techniques are discussed and results obtained by the two methods are compared.
Digital holography is widely used nowadays for interferometric studies of various objects and processes. However, peculiarities of objects under study often imply difficulties in holograms recording, reconstruction and processing. One of the major factors is a typically large number of singular points at phase distributions caused by either low signal to noise ratio at the recorded holograms or sample inhomogeneities. The basic operations applied for absolute phase extracting from digital holograms are noise filtration, phase unwrapping and subtraction of phase distributions. In this paper we demonstrate that the sequence of these operations may drastically affect the resulting image quality and the data obtained. An optimized algorithm suitable for studies of dynamic processes in biological media on microscopic level has been developed. The algorithm was applied for monitoring of nonradiative deactivation processes occurring in onion cell specimens at photosensitized generation of singlet oxygen.
The paper presents a novel combined approach aimed to detect and monitor singlet oxygen molecules in biological specimens by means of the simultaneous recording and monitoring of their deactivation dynamics in the two complementary channels: radiative and nonradiative. The approach involves both the direct registration of phosphorescence at the wavelength of about 1270 nm caused by radiative relaxation of excited singlet oxygen molecules and holographic recording of thermal disturbances in the medium produced by their nonradiative relaxation. The data provides a complete set of information on singlet oxygen location and dynamics in the medium. The approach was validated in the case study of photosensitized generation of singlet oxygen in onion cell structures.
The paper presents a novel approach to detect and monitor excited biomolecules by means of holographic registration of thermal disturbances produced by their radiationless deactivation. The photoacoustic and photorefractive methods do not provide any data on spatial distribution of these disturbances. Holographic interferometry allows one to obtain in a single shot a 2D image of the area under study containing information on spatial distribution of local variations of refractive index induced by temperature variations. The method feasibility is demonstrated on the monitoring of photosensitized generation and radiationless deactivation of singlet oxygen in water.
We present the results of theoretical and experimental studies of the polarized fluorescence in fluorophores excited by
two-photon two-color (2P2C) femtosecond laser pulses. Quantum mechanical expressions describing the fluorescence
polarization have been derived using the spherical tensor technique for asymmetric top molecules under the condition of isotropic rotation diffusion for arbitrary polarization of each of the three photons involved in the photoprocess. The expressions are presented in terms of the molecular parameters MK(R, R´, t) which contain all information about the photoprocess dynamics and can be directly determined from experiment. Ab initio computations of the flurophore structure and two-photon dynamics have been performed for p-terphenyl and indole in vacuum and in solutes using the polarizable continuum model and TD-DFT method, respectively. In case of p-terphenyl, full geometry optimization of the low-lying excited singlet electronic of the D2h symmetry has been performed. The results obtained indicate that that the solute molecules do not affect noticeably the position of the p-terphenyl energy levels which conclusion nicely fits the results reported elsewhere. In case of indole, the energy of low-lying molecular states, their permanent dipole moments, and transition dipole moments to the ground state have been computed both for non-relaxed and relaxed geometries. The results obtained manifests strong influence of the polar solute (methanol) on the position of the lowlying molecular energy levels and on the dipole moments. Using the results of the computation obtained three molecular parameters of the zero-th order, M0(0,0), M0(2,0), M0(2,2) have been calculated and compared with their values obtained experimentally. The comparison shows excellent agreement between the theory and experiment.
Oriented ground state 52S1/2 Rb atoms produced in molecular photodissociation had been observed and studied. The cell containing low-pressure RbI vapors was illuminated by pulse circularly polarized laser radiation at 266 nm. Resulting polarized Rb atoms had been detected using transmission monitoring technique in two experimental geometries. Oriented atomic spin precession in an external magnetic field was detected by atomic vapors dichroism.
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