Automatic segmentation of lysosomes and analysis of intracellular pH with Radachlorin photosensitizer and FLIM.

We report application of the fluorescence lifetime imaging microscopy (FLIM) for analysis of distributions of intracellular acidity using a chlorin-e6 based photosensitizer Radachlorin. An almost two-fold increase of the photosensitizer fluorescence lifetime in alkaline microenvironments as compared to acidic ones allowed for clear distinguishing between acidic and alkaline intracellular structures. Clusterization of a phasor plot calculated from fits of the FLIM raw data by two Gaussian distributions provided accurate automatic segmentation of lysosomes featuring acidic contents. The approach was validated in colocalization experiments with LysoTracker fluorescence in living cells of four established lines. The dependence of photosensitizer fluorescence lifetime on microenvironment acidity allowed for estimation of pH inside the cells, except for the nuclei, where photosensitizer does not penetrate. The developed method is promising for combined application of the photosensitizer for both photodynamic treatment and diagnostics.

Photophysical, rotational and translational properties of Radachlorin photosensitizer upon binding to serum albumins.

INTRODUCTION: Although photophysical properties of Radachlorin photosensitizer (PS) were extensively studied in solutions and cells, no data is available on variations of its characteristics upon binding to serum albumins, which are major transporters in blood and nutrients in cell culture media. OBJECTIVES: The primary objective of this study was to analyze changes in photophysical properties of Radachlorin molecules upon their binding to human and bovine serum albumins at different microenvironment properties. METHODS: Experiments were performed using time-resolved fluorescence spectroscopy and fluorescence recovery after photobleaching. Variations in fluorescence spectra and lifetime, fluorescence anisotropy, rotational and translational diffusion of PS molecules upon binding to albumins were studied in normal, basic and acidic conditions and at different concentrations of albumin and PS molecules. RESULTS: Radachlorin molecules effectively bind to both types of serum albumins, which causes changes in photophysical properties of the PS. A minor red shift of the fluorescence spectrum, an increase in fluorescence lifetime and anisotropy and substantial decrease of translational and rotational mobility of PS molecules were observed upon their binding to albumins. The analysis of rotational diffusion time provided robust evaluation of the bound fraction of PS molecules. Both the highly acidic microenvironment and increase in alcohol concentration above 40% resulted in detachment of PS molecules from albumins. Photophysical properties of Radachlorin in complexes with BSA and HSA were found to be slightly different. CONCLUSIONS: Binding of Radachlorin photosensitizer to either BSA or HSA affects significantly its photophysical properties, which may also vary with microenvironment acidity and alcohol concentration.

Analysis of Radachlorin localization in living cells by fluorescence lifetime imaging microscopy.

Intracellular localization of photosensitizer molecules is influential on cell death pathway at photodynamic treatment and is thus an important aspect in achieving enhanced efficacy of photodynamic therapy. In this paper we performed thorough studies of the distribution of Radachlorin photosensitizer in three established cell lines: HeLa, A549, and 3T3 with fluorescence lifetime imaging microscopy through the analysis of lifetime distributions. Experiments carried out in Radachlorin solutions in phosphate buffered saline revealed the pronounced dependence of the fluorescence quantum yield and lifetime on solution pH. This finding was used for analysis of lifetime images of living cells and their phasor plot representations and allowed us to suggest that Radachlorin localized predominantly in lysosomes, known to have acidic pH values. Experiments on co-localization of Radachlorin fluorescence lifetimes and LysoTracker fluorescence intensity supported this suggestion. The results obtained show that the inhomogeneity of fluorescence quantum yield within a cell can be significant due to lower pH values in lysosomes than in other intracellular compartments. This finding suggests that the actual amount of accumulated Radachlorin can be underestimated if being evaluated solely by comparison of fluorescence intensities.

Comparative analysis of Radachlorin accumulation, localization, and photobleaching in three cell lines by means of holographic and fluorescence microscopy.

In this paper we compare the response of cells of established lines of different origin: HeLa, A549 and 3T3 to photodynamic treatment with Radachlorin photosensitizer. The analysis was performed on different aspects of the treatment procedure including photosensitizer accumulation, localization and photobleaching in cells and post-treatment dynamics of changes in cellular morphology at different treatment doses. It was shown that in the three cell lines Radachlorin accumulated in lysosomes to much greater extent than in mitochondria. The cells' response to treatment was analyzed by identification of their death pathways and evaluation of average phase shift dynamics using digital holographic microscopy. The analysis performed on the three cell lines allowed us to evaluate treatment doses specific for each pathway in each line. Among the three lines HeLa cells were found to be the most susceptible to treatment while 3T3 cells the most resistant. The comparison of these results with the data on Radachlorin accumulation, localization and photobleaching rates showed that the observed higher sensitivity of HeLa cells to photodynamic treatment correlated with higher photosensitizer uptake and more intensive photobleaching while lower sensitivity of 3T3 cells correlated with lower uptake and less intensive photobleaching.

Singlet oxygen generation in aerosol jet and on biological surfaces.

The paper presents steady-state and time-resolved experiments on photophysical processes associated with photodynamic inactivation of infections provided by nebulization of Radachlorin photosensitizer solution. As models of surfaces subjected to photodynamic inactivation we used glass, plant leaf, mushroom cap peel and superficial fascia of chicken and salmon skin flaps. The oxygen content in the photosensitizer solution was varied by blowing with atmospheric air and with pure oxygen. It was shown that singlet oxygen was generated efficiently in the aerosol jet and that its amount increased noticeably at higher oxygen concentrations. The kinetics of photosensitizer photobleaching on different surfaces were found to be significantly different with characteristic decay times varying from seconds for leaf and glass to minutes for fascial flaps. This observation was attributed to much faster oxygen depletion on rough crumbly surfaces of biological samples due to effective oxidation reactions occurred. The singlet oxygen generation and degradation times, and the relative quantum yield were determined on different surfaces by recording time-resolved phosphorescence at about 1270 nm under normoxic and hyperoxic conditions and analyzed on the basis of the set of master equations. The results obtained provide reference marks for choosing optimal irradiation durations for photodynamic inactivation of pathogenic infectious agents (bacteria, mycobacteria, fungi, viruses) on mucous membranes, including the tracheobronchial tree.

Significant difference in response of malignant tumor cells of individual patients to photodynamic treatment as revealed by digital holographic microscopy.

The investigation of in-vitro response of cell cultures derived from tumor material of individual patients with similar tumor localizations to photodynamic treatment is presented. Tumor types included in the research were renal cell carcinoma, melanoma and alveolar, synovial, lypo- and osteo- sarcomas. Long-term observations of treatment-induced morphological changes in cells were performed by means of digital holographic microscopy. A substantial variance in response of cells of individual patients with similar tumor types and localizations to photodynamic treatment with the same dose has been observed. These peculiarities are indicative of the demand to personalized protocols of photodynamic treatment. The elevated resistance of cells of some patients to treatment at high doses highlights potential limitations of photodynamic therapy for some patients. Digital holographic microscopy is shown to be an informative label-free noninvasive tool allowing for long-term monitoring of cell samples in vitro and providing quantitative information on necrosis rate and loss of cellular dry mass. The developed methodology can be generalized for analysis of cellular response to various therapies.

In vitro monitoring of photoinduced necrosis in HeLa cells using digital holographic microscopy and machine learning.

Digital holographic microscopy supplemented with the developed cell segmentation and machine learning and classification algorithms is implemented for quantitative description of the dynamics of cellular necrosis induced by photodynamic treatment in vitro. It is demonstrated that the developed algorithms operating with a set of optical, morphological, and physiological parameters of cells, obtained from their phase images, can be used for automatic distinction between live and necrotic cells. The developed classifier provides high accuracy of about 95.5% and allows for calculation of survival rates in the course of cell death.

Quantitative assessment of changes in cellular morphology at photodynamic treatment in vitro by means of digital holographic microscopy.

Temporal dependence of changes in the morphological characteristics of cells of two cultured lines of cancer origin, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer, have been monitored using digital holographic microscopy during first two hours after short-term irradiation. The observed post-treatment early dynamics of the phase shift in the transmitted wavefront indicated several distinct scenarios of cell behavior depending upon the irradiation dose. In particular the phase shift increased at low doses, which can be associated with apoptosis, while at high doses it decreased, which can be associated with necrosis. As shown, the two cell types responded differently to similar irradiation doses. Although the sequence of death scenarios with the increase of the irradiation dose was the same, each scenario was realized at substantially different doses. These findings suggest that the average phase shift of the transmitted wavefront can be used for quantitative non-invasive cell death characterization. The conclusions made were cofirmed by commonly used test assays using confocal fluorescent microscopy.

Morphological changes in the ovarian carcinoma cells of Wistar rats induced by chemotherapy with cisplatin and dioxadet.

The development of new express methods for the analysis of the efficacy of anti-cancer therapy on the cellular level is highly desirable for the analysis of chemotherapeutic agent performance. In this paper we suggest the use of parameters of cell morphology determined by holographic microscopy and tomography for the effective label free quantitative analysis of cell viability under antitumor chemotherapy and thus of cytostatic agent efficacy. As shown, measured phase shifts and cell morphology change dramatically as a result of chemotherapy and depend strongly on the cell type and agent applied. Experimentally, a comparative analysis of the antitumor efficacy of the two cytostatics, cisplatin and dioxadet, that are commonly used for chemotherapy of disseminated ovarian carcinoma has been performed. The experiments were carried out on the Wistar rat model. An essential difference in the morphology of cells, both normal (erythrocytes) and cancerous, present in ascitic fluid taken from the non-treated group of rats and the groups treated with either dioxadet or cisplatin, has been observed. The results obtained can be interpreted as an indication of the antitumor performance of both cytostatics at the cellular level and as a demonstration of the higher efficacy of therapy with dioxadet as compared to that with cisplatin. Differences in cell morphology are suggested to be applied as quantitative markers of cell viability and cytostatic agent efficacy. The conclusions made are supported by a comparison with the results of recent experiments based on survival rates of laboratory animals treated with these agents..

Fluorescence anisotropy in indole under two-photon excitation in the spectral range 385-510 nm.

This paper presents the detailed study of two-photon excited fluorescence in indole dissolved in propylene glycol produced by two-photon absorption from the molecular ground state to several high lying excited states. The experimental method involved excitation with linearly and circularly polarized femtosecond pulses and time-resolved detection of the polarized fluorescence decay. The fluorescence intensity, anisotropy, excited state lifetime, and rotation diffusion time as function of the excitation light wavelength in the spectral range 385-510 nm were determined in experiment. The theoretical fit of the experimental results obtained demonstrated the contributions of six highly excited molecular states of different symmetry to the two-photon absorption intensity and fluorescence anisotropy. An intense two-photon absorption peak was observed experimentally in the spectral range 385-480 nm and explained as contributions from four high lying electronic excited states. The temporal dependence of fluorescence intensity in indole was satisfactory characterized by a single excited state lifetime τf and a single rotational diffusion time τrot. As shown, the excited state lifetime τf depends on the excitation light wavelength, which was explained by taking into account nonradiative relaxation transitions in the molecular vibronic excited states. The rotation diffusion time τrot was found to be equal to τrot = 0.9 ± 0.5 ns and practically independent of the excitation wavelength. The determined molecular anisotropy changed substantially in the spectral area 385-480 nm taking positive and negative values, and the anisotropies referring to linearly and circularly polarized excitation light changed almost in opposite phases with each other. The experimental results obtained were interpreted using ab initio molecular structure computations and a model based on the Frank-Condon approximation and taking into account vibronic absorption bands.

Time-resolved spectral analysis of Radachlorin luminescence in water.

We report results of spectral- and time-resolved study of Radachlorin photosensitizer luminescence in water in the spectral range of 950-1350nm and for determination of the photosensitizer triplet state and the singlet oxygen lifetimes responsible for singlet oxygen generation and degradation. At any wavelength within the explored spectral range the luminescence decay contained two major contributions: a fast decay at the ns time scale and a slow evolution at the µs time scale. The fast decay was attributed to electric dipole fluorescence transitions in photosensitizer molecules and the slow evolution to intercombination phosphorescence transitions in singlet oxygen and photosensitizer molecules. Relatively high-amplitude ns peak observed at all wavelengths suggests that singlet oxygen monitoring with spectral isolation methods alone, without additional temporal resolution can be controversial. In the applied experimental conditions the total phosphorescence signal at any wavelength contained a contribution from the photosensitizer triplet state decay, while at 1274nm the singlet oxygen phosphorescence dominated. The results obtained can be used for optimization of the methods of singlet oxygen monitoring and imaging.

Digital holographic microscopy in label-free analysis of cultured cells' response to photodynamic treatment.

A methodology providing noninvasive monitoring and evaluation of the effect of photodynamic treatment on live cells in vitro is presented. Variations in morphological characteristics of cells in the course and after treatment are recorded by means of digital holographic microscopy. High-precision measurements of phase shift gained by probe radiation in HeLa and human endometrial mesenchymal stem cell cultures demonstrate for the first time changes of their volume occurred in response to treatment.

Femtosecond Time-Resolved Photofragment Rotational Angular Momentum Alignment in Electronic Predissociation Dynamics.

This Letter presents an experimental and theoretical study of femtosecond time-resolved vector correlations in methyl iodide (CH3I) electronic predissociation via the second absorption B-band at 201.2 nm. The time evolution of the phenomenological anisotropy parameters ßl was determined from time-resolved photofragment angular distributions obtained by means of the femtosecond laser pump-probe technique coupled with velocity map imaging detection of vibrational ground-state CH3(ν = 0) fragments and spin-orbit excited I*(2P1/2) atoms. Theoretical interpretation of the experimental results was performed on the basis of a fitting procedure using quasiclassical theory, which elucidates vector correlations in photodissociation of symmetric top molecules. The results of the fitting are in very good agreement with the experimental data and demonstrate the important role of molecular excited-state lifetimes, parent molecule and methyl fragment rotations, and methyl fragment angular momentum alignment on the time-dependent electronic predissociation dynamics.

Dynamics of two-color two-photon excited fluorescence of p-terphenyl: determination and analysis of the molecular parameters.

We present the experimental and theoretical study of the two-photon excited polarized fluorescence of p-terphenyl dissolved in cyclohexane/paraffin. The fluorescence was produced within a two-color two-photon (2C2P) excitation scheme utilizing simultaneous absorption of two femtosecond laser pulses at 400 nm and at 800 nm with the total excitation energy of 4.649 eV. The fluorescence was detected by a time correlated single photon counting (TCSPC) system with two detectors. Using different combinations of the absorbed photon polarizations we extracted seven time-dependent molecular parameters from experiment that contain all information on the dynamics of the three-photon process under study. The analysis of the obtained molecular parameter values was based on the ab initio calculations of the vertical excitation energies and transition matrix elements in p-terphenyl and allowed for determination of the whole structure of the two-photon absorption tensor, fluorescence lifetime, and the rotational correlation time. The obtained results imply that the fluorescence in the conditions of our experiment was governed mostly by the d(z) component of the fluorescence transition dipole moment that is parallel to the molecular long axis Z. The tensor was found to be symmetric. The two-photon excitation in p-terphenyl occurs simultaneously via two channels, one of them resulting in the population of the totally symmetric excited state and the other in the population of the nontotally symmetric excited state. Moreover, the energetically allowed pure electron transitions are dipole forbidden and become allowed by vibronic coupling.

The role of intermediate state polarization in determination of vector properties of the ground state using multiphoton excitation.

We present a general theory for calculating the vector and geometrical properties of the multiphoton excitation of an arbitrary atomic or molecular system. The results are applied to study the influence of the polarization of the two-photon excited state, which is usually neglected, on the intensity of (2 + 1) resonant multiphoton ionization in atoms. Two examples of specific atomic systems of practical importance are presented: oxygen and chlorine. For some cases, the effect of the polarization of the pre-ionized state can be significant and must be properly treated.

Intermediate state polarization in multiphoton ionization of HCl.

The paper presents the detailed theoretical description of the intermediate state polarization and photofragment angular distribution in resonance enhanced multiphoton ionization (REMPI) of molecules and the experimental investigation of these effects in the E(1)Sigma(+) and V(1)Sigma(+) states of HCl populated by two-photon transitions. It is shown that the intermediate state polarization can be characterized by the universal parameter b which is in general a complex number containing information about the symmetry of the two-photon excitation and possible phase shifts. The photofragment angular distribution produced by one- or multiphoton excitation of the polarized intermediate state is presented as a product of the intermediate state axis spatial distribution and the angular distribution of the photofragments from an unpolarized intermediate state. Experiments have been carried out by two complementary methods: REMPI absorption spectroscopy of rotationally resolved (E,v'=0<--X,v"=0) and (V,v'=12<--X,v"=0) transitions and REMPI via the Q(0) and Q(1) rotational transitions followed by three-dimensional ion imaging detection. The values of the parameter b determined from experiment manifest the mostly perpendicular nature of the initial two-photon transition. The experimentally obtained H(+) -ion fragment angular distributions produced via the Q(1) rotational transition show good agreement with theoretical prediction.

Spin-polarized hydrogen atoms from molecular photodissociation.

The production of spin-polarized hydrogen atoms from the photodissociation of hydrogen chloride with circularly polarized 193-nanometer light is inferred from the measurement of the complete angular momentum distributions of ground state Cl(2P3/2)and excited state Cl(2P1/2)cofragments by slice imaging. The experimentally measured and ab initio predicted a q(k) (p)parameters, which describe the single-surface and multiple-surface-interference contributions to the angular momentum distributions, are in excellent agreement. For laser pulses longer than about 0.7 ns, the polarization of the electron and the proton are both 36%.