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..

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.