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.
•Variations of Radachlorin fluorescence lifetime in cells provide data on acidity.•Phasor plot clusterization allowed for automatic segmentation of acidic structures.•Automatically segmented acidic clusters coincided with LysoTracker fluorescence.
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.
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•Radachlorin fluorescence lifetime and quantum yield vary with microenvironment pH.•Radachlorin fluorescence lifetimes differ in different cell compartments.•Radachlorin localizes ...predominantly in lysosomes.•Changes of Radachlorin fluorescence lifetimes with pH are reversible.
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.
•Different cell lines demonstrate different sensitivity to photodynamic treatment.•Radachlorin mostly accumulates in lysosomes in HeLa, A549 and 3T3 cell lines.•HeLa cells show higher PS accumulation ...and higher bleaching rate than A549 and 3T3.
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.
Changes in morphological and optical parameters of HeLa cells preincubated with 5-aminolevulinic acid (5-ALA), after photodynamic treatment (PDTr) with different intensity of excitation radiation are ...studied by digital holographic microscopy. Optimization of the algorithm of cell segmentation on their phase image is performed. The developed algorithm allows for automatic data acquisition on extended amounts of cells in a sample (over 600). The morphological and optical parameters of cells at different doses of PDTr were determined, and the lognormal distribution of average phase shift of the cells was demonstrated. Changes in the cell parameters at different radiation doses which initiate programmed cell death and necrosis were recorded.
Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic ...chemistry. We have designed and synthetized four novel iridium Ir(N^C)
(N^N)
complexes (N^C-benzothienyl-phenanthridine based cyclometalated ligand; N^N-pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters' solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry. The complexes exhibited excitation close to the biological "window of transparency", NIR emission at 730 nm, and quantum yields up to 12% in water. The compounds with higher degree of the chromophore shielding possess low toxicity, bleaching stability, absence of sensitivity to variations of pH, serum, and complex concentrations. The properties of these probes as oxygen sensors for biological systems have been studied by using phosphorescence lifetime imaging experiments in different cell cultures. The results showed essential lifetime response onto variations in oxygen concentration (2.0-2.3 μs under normoxia and 2.8-3.0 μs under hypoxia conditions) in complete agreement with the calibration curves obtained "in cuvette". The data obtained indicate that these emitters can be used as semi-quantitative oxygen sensors in biological systems.
In the prevailing era, the most relevant issues seem to be the development of an adaptive policy of an energy sales company to ensure financial stability and sustainable development in the course of ...energy sales activities. This study continues the cycle of the author's general
research devoted to the methodological issues of an adaptive policy development in relation to the key business processes of energy sales activities implemented by an energy sales company, the specifics of its work as a guaranteed energy (capacity) supplier, etc. The article examines the conditions and factors that determine the need to develop an adaptive system for an energy sales company to organize payment acceptance. When it comes to the business process organization for payment acceptance, energy retail companies need to take into account the conditions and factors that set the long-term trend in this area: changes in legislation, the strength of market competition and the trends of the payment acceptance market infrastructure, the ongoing coronavirus infection pandemic, and the ongoing technological change. Management decision-making should be based on the principles of economic feasibility and efficiency in conjunction with legal requirement compliance. Power supply companies need to use the conditions and factors highlighted in the study to build up competitive advantages in the field of payment acceptance organization, to maintain and develop their market position, which can only be done through an adaptation policy in this area. For the purpose of economic efficiency increase concerning organization payment acceptance business process, energy sales companies need to plan the indicators of the payment acceptance system, implement measures for their implementation (achieve target values), carry out the analysis of the achieved results and their reflection, which will ensure timely and reasonable acceptance of management decisions.
CdSe and CdTe-based semiconductor fluorescent nanocrystals, also called quantum dots (QDs), attract the attention of biologists due to their wide range of emission in a visible light interval, high ...fluorescence quantum yield and photostability. However, their application is limited because of possible toxicity of cadmium. Indeed, there is a probability of metal leakage from QDs cores as a result of damage of both inorganic and organic layers of shells covering QDs. An alternative to cadmium QDs could be nanostructures having as a core, for example, non-toxical indium phosphide (InP), also emitting in the visible region of the spectrum. At present, there is few works on the use of these particles in biology. In this study, a comparative analysis of the spectral-luminescent properties of two InP/ZnS-QDs samples coated with PEG carrying- COOH or -NH
2
functional groups was performed. The obtained data were compared with the characteristics of CdSe/ZnS-QDs coated with PEG. The photophysical properties of all QDs in aqueous solution corresponded to the information claimed by manufacturers, but the fluorescence quantum yield of InP-based nanoparticles was found to be lower than that of CdSe-QDs. We also show that the photoluminescence of all types of QDs at pH 4.0 was lower than at pH 7.4, while the decrease in fluorescence intensity was minimal in the case of QDs-PEG-COOH. Studying the uptake of all three types of QDs by J774 macrophages, we found that the fluorescence spectra of internalized QDs do not change in comparison with those in solution. All three types of QDs after 24 hours of incubation were accumulated in the cells, but while QDs-NH
2
and QDs without reactive groups were detected mainly in vesicular-like discrete structures, the QDs-COOH were diffusely distributed throughout the cytoplasm. This fact indicates different mechanisms of interaction with cell membranes. In nonphagocytic HeLa cells all types of QDs behaved similarly, but the overall level of cells fluorescence was much lower. This may be due to both reduced nonspecific uptake and possible quenching of QDs fluorescence in acidic endolysosomes. Cytofluorimetric analysis of propidium iodide accumulation showed that after 24 hours incubation with all studied types of QDs as well as in control (no QDs), the proportion of dead HeLa cells did not exceed 10%. Thus, it has been demonstrated that non-toxic InP-based QDs can be used as an effective tool for biological research.
The article concentrates on development of methodological tools for creating a model of cluster structures sustainable development in shipbuilding of Russia through the example of the shipbuilding ...cluster of the Primorye Territory. The proposed tools can complement the existing methods for clustering industries and regions in business processes scientific and professional sphere. The proposed method of assessing regional economic system availability for clustering and algorithm for its implementation allowed to identify risks and limitations of the regional shipbuilding cluster sustainable development. The results of the research are: allocation of target structure on the cluster strategic map in terms of a balanced system of indicators, manifestation of in-system characteristics of the cluster structure model, definition of key directions for development of cooperation and collaboration relations. Further, the prerequisites were established for development of network interaction, cooperation and collaboration between the participants of the shipbuilding cluster and the parties involved in Russia and Asia-Pacific countries. The author's contribution to the development of scientific knowledge in problems of clustering and professional sphere is formulation and manifestation of in-system components of cluster structure business model, initiation of target structure of their statistical, controlling, dynamic and predictive changes, which makes it possible to form monitoring systems at the stages of a cluster life cycle and ensure timely managerial action. The approach proposed by the author allows to reduce risks of uprising institutional trap and management disorders in the decisionmaking process on cluster-network structures establishment and functioning.
The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various ...pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 μm using PLIM and showed a gradient in the intracellular O2 level towards their center.
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