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 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.
We present validation of a tilt angle illumination technique for height maps’ reconstruction using digital holography and propose a few solutions for improvement of the inclined probe wave front approach. The general algorithm of tilt angle digital holography approach is described and validated in experimental monitoring of a coin relief. Several approaches of height map quality improvement concerning tilt angle estimation, relation between measurement accuracy and dynamic range, and selective averaging algorithm are proposed and discussed.
This work further develops a recently proposed time-resolved inline digital holography (TRIDH) [Petrov, N. V. et al. Opt. Lett. 43, 3481 (2018)] for studying degenerate phase modulation induced by an inclined collimated pump beam in the glass substrate with the quantum dots at the surface. Similar to many techniques for measuring nonlinear properties of materials, it is based on a comparison of the prediction obtained by the mathematical model of the phenomenon with experimental data. We have extended the mathematical model for the case of interaction of two femtosecond laser pulses in the double-layered sample. The impact of the ratio between nonlinear refractive indexes of two layers and their thicknesses on induced phase modulation is analyzed.
Complex wavefront manipulation is a promising technique for many applications of optics and photonics. In this report we will present our results on development of DMD wavefront correction experimental setup and discussion of its performance for various parameters of binary fringe pattern and 1st diffraction order filtration aperture. It was shown that trade-off between spatial resolution and discretisation of the desired amplitude and phase distribution should be achieved. Decrease of the binarized interference fringes width results in higher spatial resolution of the modulated complex wave but increase discretisation of amplitude and phase distributions as well. The correction of wavefront aberrations using digital micromirror device was performed. We observed significant reduction in wavefront phase error by conduction of Zernike polynomials decomposition.
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.
In this work we demonstrate wavefront complex modulation of semiconductor light sources via digital micromirror device (DMD). Proposed holographic configuration allows to correct the aberrations caused by the imperfections of the DMD and the optical elements. We consider the blazing effect of the DMD surface and configured the optimal condition of the optical setup for certain DMD and the wavelength of the radiation source. The technique was approved with the experiment of obtaining different kinds of wavefront distributions from the semiconductor laser with high M2 factor.
One of the problems of interferometric methods is the difficulty of measuring surface shape with sharp boundaries due to the wavelength-limited dynamic range of the measurement. To circumvent this limitation multiwavelength methods or techniques based on hologram capturing at the different tilt of the illumination beam are applied. In this work we examine the performance of the digital holographic interferometry with multi-inclination illumi- nation in the numerical and real experiments. Lensless implementation of the technique implies the wavefront propagation by numerical algorithms. In this regard the speckle scattering in the Fresnel diffraction area caused by surface roughness and the impact of distance from the object to the registration plane are analyzed. Since shape measurement is based on the calculation of phase difference for the wavefronts recorded with tilt of the object illuminating beam, the requirements to preciseness of measurements of the angle of incidence of this beam are considered. The algorithm of the inclination angle determination are developed. The performance of noise suppression techniques, namely sine-cosine and BM3D methods are considered for high noisy conditions, when the phase distributions are formed by reflecting object with a great roughness and height differences.
In this paper we present a comprehensive description of the pump-probe holographic arrangement and data processing procedure optimized for reconstruction of long smooth strain wave patterns in transparent solids. The approach was tested on detection of nonlinear strain waves generated in a uniform PMMA bar by initial shock pulses of different energies. Phase images representing these waves in the bulk of the waveguide are demonstrated. The strain wave parameters and evolution were shown to be substantially dependent on the initial shock pulse amplitude.
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.
We demonstrate an alternative approach to determination of the third order elastic moduli of materials based on
registration of nonlinear bulk strain waves in three basic structural waveguides (rod, plate and shell) and further
calculation of the Murnaghan moduli from the recorded wave parameters via simple algebra. These elastic moduli are
available in literature for a limited number of materials and are measured with considerable errors, that evidences a
demand in novel approaches to their determination.
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.
Modern structural elements are often made of laminated polymer materials or composites on the base of polymer matrices. The proper functioning of these elements may be of vital importance especially in automotive and aerospace industries, in gas and oil transportation. The major problem in their performance is a possibility of a sudden and irreversible delamination caused by various factors. We propose and study a NDT approach aimed to detect delamination areas in adhesively bonded layered structural elements made of different materials. The proposed approach is evaluated by use of holographic detection and monitoring of the evolution of bulk strain solitons generated in such structures.
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.
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