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.