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Abstract
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.
© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
A persistent increase of cancer incidences and high recurrence rates are key problems in modern oncology requiring improvement of existing treatment modalities and development of novel approaches for early diagnostics and therapy. One of highly promising modalities is photodynamic therapy (PDT) which was already successfully applied for treatment of various malignant and benign pathologies, skin lesions, macular degeneration, microbial infections etc. [1–3]. PDT employs specific physical-chemical properties of molecular photosensitizers (PSs) which are known to be selectively accumulated in pathological tissues with enhanced metabolism. Excitation of PS molecules by light within an absorption band leads to the formation of reactive oxygen species (ROS) that can cause cell death, tumor resorption and ablastics of the lesion.
The anti-tumor effect of PDT is provided by the three interconnected processes: direct death of tumor cells, vascular disruption and activation of immune response [4]. Although the integral effect of PDT is well established, contributions and mechanisms of occurring specific intracellular processes are still far from being sufficiently understood [5,6]. Moreover it was presumed that the contribution of each of these processes to the tumor response to PDT may vary depending upon the tumor localization [7], PS type, PDT protocol parameters, irradiation dose and duration. The analysis of individual cells’ response to photodynamic (PD) treatment at various conditions is thus an essential aim for the study of PS efficacy and evaluation of optimal treatment doses.
A considerable progress has recently been achieved in characterization of cell death mechanisms and pathways. The commonly used classification of cell death through apoptosis and necrosis has been added by more subtle distinction, and several novel mechanisms of cell death have been introduced, see e.g. [6,8,9]. The Nomenclature Committee on Cell Death has recently released definitions for four typical (apoptosis, necrosis, autophagy and cornification) and eight atypical mechanisms of cell death [10]. Moreover a cell can even switch back and forth between different death pathways [11].
From the viewpoint of present-day research on cellular response to PD treatment the reliable distinction between apoptotic and necrotic pathways along with determination of corresponding treatment doses is still highly desirable. While necrosis is considered as a quick, violent and unprogrammed cell death caused by excessive chemical or physical impact, apoptosis is a cell suicide developing in accordance to a specific program. Necrosis is characterized by cytoplasm swelling, organelles destruction and plasma membrane disintegration, which results in the efflux of intracellular contents, causing in vivo inflammation. On the contrary, apoptosis involves cell shrinkage, more tight packing of cytoplasm and organelles, that is followed by extensive plasma membrane blebbing and formation of separate apoptotic bodies which are in vivo phagocytised by macrophages or adjacent normal cells [12,13]. Determination of treatment doses providing cell death mainly through apoptosis is highly desirable for medical implementations since it is less harmful for patients and does not cause further inflammatory reaction.
The distinction between apoptotic and necrotic behavior of cells is usually made by assessment of cell membrane integrity using specific standard test assays and further analysis by confocal fluorescent microscopy. Other approaches are based on determination of morphological changes of cells by means of flow cytometry or light and transmission electron microscopy (TEM). Note that until quite recently TEM was considered a “gold standard” to confirm apoptosis [12]. However most of these methods (TEM microscopy, flow cytometry) do not allow for monitoring cellular changes in dynamics, they rather provide information on cell condition in a certain time period. Besides that fluorescence-based techniques necessarily require specific fluorescent probes which can alter cellular characteristics.
Optical techniques operating with phase variations of the radiation passed through the object are nondestructive and allow for monitoring cellular changes in dynamics. The oldest method from this group is phase contrast microscopy, still widely applied in cellular research. This method allows for recording cell images without staining and with higher image contrast than regular light microscopy. However quantitative description of cellular morphology is problematic with this technique. Much more informative are techniques of quantitative phase imaging, digital holography in particular, which have important advantages and already found wide applications in research of various processes at the cellular level (see e.g. [14–19]). We have recently reported [20] our first results on determination of cellular morphology at PD treatment by means of digital holographic microscopy. The necrotic pathway of cell death was investigated in two types of cell cultures: HeLa and mesenchymal stem.
In this paper we present results of thorough monitoring of morphological characteristics of cells of the two widely used cultured cancer cell lines, HeLa and A549, in the course and during 60-90 minutes after PD treatment with chlorin PS at various irradiation doses. The monitoring was carried out by means of digital holographic microscopy. High-precision measurements of phase shift gained by probe radiation in targeted cells demonstrated changes of their volume in the course and after PD treatment. The phase shift dynamics has been analyzed as function of treatment parameters. The post-treatment dynamics of phase shift demonstrated several distinct scenarios of cell death depending upon the irradiation dose. In particular the methodology applied allowed for clear separation between apoptosis and necrosis. The experiments performed by digital holographic microscopy were assisted by observations of the same cells by far-field microscopy. The cell membrane integrity was examined by the commonly used Acridine Orange and Ethidium Bromide (AO/EB) test assay with observations of its fluorescence using confocal fluorescence microscopy. The apoptotic pathway of cell death was confirmed using the Annexin-V and Propidium Iodide (AnnexinV/PI) test assay.
2. Experimental approach
1 Specimen preparation
Investigations of living cells’ response to photodynamic treatment were performed on two cultured cell lines: human cervix epidermoid carcinoma HeLa cells and human alveolar basal epithelial adenocarcinoma A549 cells (both from the Russian Cell Culture Collection, Institute of Cytology RAS, St. Petersburg, Russia). Cells were cultivated in the Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37$^o$C in 5% CO$_2$ atmosphere. In 48 h after seeding on Petri dishes Radachlorin PS (RadaPharma, Russia) was added to the culture medium at final concentration of 5 $\mu$g/ml. Cells were incubated in this solution for 4 hours, then the medium was replaced by that without PS. Radachlorin comprises a composition of sodium salts of chlorin e6 ($\sim 80\%$), purpurin 5 ($\sim 15\%$) and chlorin p6 ($\sim 5\%$). As shown in [20,21] this PS penetrates through the cellular membrane and accumulates mainly in mitochondria, lysosomes and endoplasmic reticulum. Basic photophysical properties of Radachlorin in aqueous solutions were studied in detail in our recent works [22–24], where the efficient generation of singlet oxygen was demonstrated.
PS-loaded cells were irradiated by a diode laser operating at 660 nm, close to the maximum of the Q absorption band of the chlorin PS. The laser beam fluence rate was varied within the range of 6-130 mW/cm$^2$. To provide nondestructive monitoring of cell parameters the holograms recording was carried out using a low-power CW HeNe laser operating at 633 nm, outside the PS absorption bands. The recording radiation fluence rate was maintained at about 50 $\mu W/cm ^2$.
2 Digital holograms recording and processing
Changes of cellular morphology resulting from PD treatment were monitored by means of an inverted digital holographic microscope in the off-axis Mach-Zehnder layout (see [20] for details). A 20x microscope objective and a collimating lens in the object channel provided spatial resolution of about 0.8 $\mu m$. Relatively low magnification allowed us to observe several cells at each phase image. Automatic scanning of the sample was performed using a two-coordinate motorized stage (Standa); holograms were recorded by a Videoscan-205 CCD camera (Videoscan) controlled by a software designed in the LabView 8.5 development system. Iterative monitoring of a set of 7x7 specimen areas within the irradiated spot was carried out at room temperature every five minutes during 1.5 hours after irradiation. This procedure allowed to ensure data robustness and reproducibility. To increase the quality of obtained phase images several digital holograms were recorded at each position of the motorized stage and the one with the highest contrast was used for reconstruction. Hologram quality was assessed by calculation of the total intensity in the 1-st diffraction order. This allows obtaining high-quality phase images even in conditions of minor vibrations, caused by motorized stage movement and other sources. Figure 1 demonstrates representative 3D phase plots of HeLa cells obtained before and after PD treatment. Some noise noticeable in phase plots is due to coherent nature of the probe laser radiation and its low intensity necessary for noninvasive monitoring of photosensitized cells. However since further determination of cellular parameters assumed data averaging over an entire cell area, it resulted in essential reduction of noise impact on the results obtained.
Fig. 1. 3D pseudocolored phase plots of HeLa cells obtained before photodynamic treatment (a, c) and in 60 minutes after irradiation at 22.1 mW/cm$^2$ (b) and 93 mW/cm$^2$ (d).
Each set of digital holograms recorded at different specimen areas contained also an image of an area without cells. This phase distribution, below referred to as a background phase image, was subtracted from each reconstructed phase distribution for compensation of optical system aberrations. Reconstruction of the recorded digital holograms was performed by means of the least square estimation algorithm [25,26] based on the assumption of slowly varying phase and amplitude distributions of the object wave. For biological objects, living cells in particular, this assumption is almost always fulfilled due to smooth shapes and high transparency of cells. An additional filtration of the obtained phase images was performed for images with high values of shot or coherent noise. Since in this research high spatial resolution was not required, smoothing of phase images was done by means of the sin-cos algorithm [27]. Afterwards, the obtained phase distributions were unwrapped using Goldstein algorithm [28]. So far as phase shift is a relative value, each unwrapped phase image was normalized to make sure that image areas containing no cells induce an approximately zero phase shift. At the final step of phase image processing cells segmentation was performed. In order to increase quality of the obtained data and to ensure correct calculation of cellular parameters the manual segmentation of individual cells at each moment of time was carried out.
3 Assessment of cellular morphology
Basically the phase shift $\delta \phi$ introduced by a cell to the transmitted wave front in a certain point of the cross-section, normal to its propagation direction, is described by the refractive index n and cell thickness l: $\Delta \phi$ $\sim \int ndl$. If the intracellular refractive index does not vary significantly, the recorded phase image of a cell characterizes spatial distribution of cell thickness. The value of phase shift averaged over entire cell is a more robust parameter since it is less sensitive to a random noise:
where $S_{cell}$ is a cell projected area. The value of total phase shift integrated over the entire cell and multiplied by a constant coefficient provides information on cellular dry mass DM [29]: where $\lambda$ is the probe laser wavelength and $\alpha$ is a refractive index increment related to the intracellular content, $\alpha \approx 0.0018 - 0.0021 m^3 /Kg$ [29] for most of cells.Therefore the average phase shift can be interpreted as a ratio of the cell dry mass and its projected area (dry mass density). A procedure for calculation of major optical and morphological parameters of cells from phase shift distributions obtained by digital holographic microscopy was described in detail by Girshovitz and Shaked [30]. Note that although several other optical and morphological parameters of individual cells can be determined from their phase images, we believe that monitoring of these parameters does not provide any additional information on cell death pathway and dynamics and is less meaningful. For example variations of cellular dry mass can be used for identification of cells necrosis only. The integrity of cellular membrane during apoptosis results in dry mass invariance.
As known, cultured cells are characterized by heterogeneity due to several reasons, such as asynchronous cell cycle and difference in individual cell shapes. In the conditions of our experiments these features resulted in significant variations of initial phase shift induced by individual cells and of their response to PD treatment. Therefore several dozens of cells were monitored in each experiment for determination of statistically significant mean values of phase shift in each cell sample. Statistical analysis of the data obtained provided robust information on the typical cellular response to PD treatment. The measurement accuracy was improved by monitoring morphological changes of the same cells at each time point during the experiment. A significant decrease of potential errors due to diversity of initial cellular parameters was thus achieved.
The conclusions made based on holographic measurements were supported by standard test assays in a scanning confocal fluorescence microscope Leica TCS SP5 and with further analysis. Cell morphology and integrity of plasma and nuclear membranes was analyzed by intravital staining with Acridine Orange and Ethidium Bromide (AO/EB) assay (Merck, Germany). According to Ribble et al [31], AO permeates cellular membranes and accumulates in acidic structures and nucleus, staining the latter green. EB penetrates into cells only if plasma membrane is disintergated and then stains the nucleus red. Since the effect of EB dominates over that of AO, live and early apoptotic cells normally have green nuclei, while dead necrotic cells normally have orange nuclei.
Cells were stained before and after PD treatment with a mixture of 2.5 $\mu$g/ml AO and 5 $\mu$g/ml EB for 1 minute. Apoptotic cells were revealed using a FITC-Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI (Thermo Fisher Scientific, USA), for Flow Cytometry, using a protocol for confocal microscopy. In both tests fluorescence of AO/EB and AnnexinV/PI was excited by Ar laser at 488 nm and was recorded in two spectral ranges: 500-560 nm for AO and AnnexinV and 590-680 nm for EB and PI. Single z-projections and z-stacks taken with 0.5-$\mu$m pitch were recorded and analyzed using ImageJ software (NIH, USA).
3. Experimental results
Our control experiments performed on 6-hour continuous holographic monitoring of photosensitized cells but not subjected to irradiation by excitation laser diode at 660 nm, demonstrated no significant changes of cellular morphology, average phase shift or dry mass (see Fig. 2(d)). Note that the probe laser power density used in holographic setup was only about 50 $\mu$W/cm$^2$ and the laser wavelength was out of the PS absorption bands.
Fig. 2. Average phase shift dynamics in HeLa (a) and A549 (b) cells at the indicated irradiation doses. (c) HeLa cells dry mass dynamics at indicated irradiation doses.(d) average phase shift dynamics in photosensitised but not irradiated HeLa cells during 6 hours of their continuous monitoring. (e) Schematics of average phase shift variation scenarios as function of fluence rate for the two cell lines. Colors in (e) correspond to those on the graphs in (a, b, c).
The dynamics of average phase shift in living cells exposed to PD treatment at various doses was monitored during 1.5 hours after short-term irradiation. The treatment dose was varied by changing the irradiation fluence rate at the same irradiation duration (5 minutes throughout all experiments). The phase shift dynamics in the two types of cells at different irradiation doses are shown in Figs. 2(a) and 2(b). Each experimental point on the graphs in Figs. 2(a) and 2(b) represents the value averaged over 50 cells and error bars indicate standard error deviation. These data were compared with the results of fluorescence analysis using standard test assays shown in Fig. 4.
Four substantially dissimilar scenarios of cells’ response at different irradiation fluence rates have been distinguished for both cell cultures.
- 1. At very low fluence rates (6.2-10.6 mW/cm$^2$ for HeLa and 15 mW/cm$^2$ for A549 cells) no significant changes in phase shift, dry mass and projected area were observed. Typical phase images of HeLa and A549 cells taken before and after PD treatment at these fluence rates are shown in the top row of Fig. 3. Fluorescent images obtained with the AO/EB test assay did not reveal any noticeable difference in cellular morphology between control, non-treated cells and PS-loaded cells irradiated at these fluence rates.
- 2. Low fluence rates (15-22.1 mW/cm$^2$ for HeLa and 46 mW/cm$^2$ for A549 cells) resulted in a slow increase of average phase shift up to a plateau level (1.2 rad for HeLa and 1.4 rad for A549 cells). The phase shift rise started in 10-15 minutes after irradiation and was observed only in a part of the monitored cells. The phase shift increase was accompanied by the decrease of cell projected area while the cellular dry mass remained unaltered. The average value of cellular dry mass determined in HeLa cells in 70 minutes after irradiation was found to be 248 pg that is very close to the initial value of 253 pg (Fig. 2(e)). Typical phase images of HeLa cells taken before and after treatment at these doses are shown in Figs. 1(a) and 1(b) and in the middle row of Fig. 3. Corresponding confocal fluorescent images taken with AO/EB staining assay (Fig. 4(d)) and AnnexinV/PI assay (Fig. 4(e)) demonstrate an absence of EB and PI fluorescence, that evidences cellular membrane integrity, while fluorescence of Annexin-V is indicative of its binding to phosphatidylserine at the outer surface of cellular membranes, that is a marker of cell apoptosis. These results are in a good agreement with the invariance of cellular dry mass confirmed by digital holography. The phase contrast image (Fig. 4(f)) also demonstrates morphological changes typical for apoptotic pathway of cell death: cell rounding and blebbing.
- 3. Further increase of fluence rate up to 38 mW/cm$^2$ for HeLa and 93 mW/cm$^2$ for A549 cells led to a quite different trend of average phase shift evolution. The delayed decrease of phase shift was observed starting in 25 min after irradiation in HeLa and in 40 min in A549 cells. Moreover, a decrease of cellular dry mass and increase of projected area were observed in both kinds of cells. In HeLa cells the dry mass decrease measured at the end of the observation time amounted to $\approx 80 pg$ (30% of the initial value).
- 4. At high fluence rates (62-93 mW/cm$^2$ for HeLa and 130 mW/cm$^2$ for A549 cells) a prominent decrease of the average phase shift and increase of the projected area were observed starting right after or even in the course of irradiation. The decrease in more than 0.06 rad during the first five minutes was recorded in HeLa cells. The considerable decrease of the average phase shift achieving a plateau at the level of 0.6-0.7 rad was observed in both types of cells. Typical phase images of HeLa and A549 cells obtained before and after irradiation at these fluence rates are demonstrated in the bottom row of Fig. 3. The observed phase shift dynamics was accompanied by a substantial reduce of cellular dry mass, for HeLa cells it comprised $\approx 100 pg$ (40% of the initial value), see Fig. 2(c). Fluorescent images of cells obtained with AO/EB staining (Fig. 4(g)) demonstrate most of cell nuclei being stained by Ethidium bromide, that is indicative of fatal disintegration of cellular membranes. Fluorescent images of cells obtained with AnnexinV/PI staining (Fig. 4(h)) show strong fluorescence of propidium iodide and minor spots of Annexin-V which also indicates membrane rupture featuring necrotic cell death. The phase contrast image (Fig. 4(i)) demonstrates collapse of the plasma membranes and lysis of the cytoplasm and organelles.
Fig. 3. Typical 2D phase images of HeLa and A549 cells before and after PD treatment. Irradiation doses are indicated on the left of each pair of images.
Therefore, as can be seen in Fig. 2(e), the scenarios 1-4 occur at different irradiation doses for the two cell lines examined. Note that within the cell response scenarios 2-4 indicated above the phase shifts recorded at the end of the observation time differed reliably from each other with the level of significance p$<$0.05.
4. Discussion
The results obtained can be interpreted in terms of different pathways of cellular response initiated at different doses of PD treatment. The tendencies observed are in general similar for both types of cells however specific trends occur at substantially different treatment doses.
The invariance of average phase shift and cellular dry mass at very low irradiation doses (shown by cyan and blue curves in Figs. 2(a) and 2(b)) indicates no cellular response to the treatment. This conclusion was confirmed by fluorescent images where no cell changes were observed. This cell behavior can be explained by low amounts of generated reactive oxygen species that can be successfully deactivated by intracellular antioxidant mechanisms and by the threshold nature of cell response to PDT.
The increase of average phase shift at higher irradiation doses, shown by green curves in Figs. 2(a) and 2(b) and by phase images in Figs. 1(a) and 1(b) and in the middle row in Fig. 3, along with the invariance of cellular dry mass and total phase shift can be explained by the decrease of cell projected area and cell rounding. This explanation is supported by the fluorescent images in Figs. 4(d) and 4(e) and phase contrast image in Fig. 4(f) demonstrating cells rounding, blebbing, membrane integrity and Annexin staining of outer leaflet of plasma membranes. All these features suggest that apparently cells became unable to withstand the impact of generated ROS and the pathway of programmed cell death through apoptosis was activated. Note that similar variations in phase images of living cells caused by other factors have recently been reported by Kemmler et al [32] and by Kemper et al [33] and referred to as early apoptosis.
Fig. 4. Images of HeLa cells subjected to PD treatment at different irradiation doses. Images of cells before irradiation (a-c) and in 50 minutes after irradiation at the fluence rates of 15 W/cm$^2$ (d-f) and 62 mW/cm$^2$ (g-i). Left column: AO(green)/EB(red) fluorescent images; middle column: Annexin-V(green)/PI(red) fluorescent images; right column: phase contrast images.
The dramatic decrease of the phase shift at high irradiation doses, shown by red curves in Figs. 2(a) and 2(b) and phase images in Figs. 1(c) and 1(d) and in the bottom row in Fig. 3, being accompanied by the decrease of cellular dry mass is indicative of cell death through necrosis. The cellular dry mass value is related to the amount of intracellular content and at normal conditions should not decrease rapidly, when averaging over several dozens of living cells. A significant decrease of this value can be explained by cellular membrane rupture and efflux of intracellular content. The process is accompanied by the decrease of total phase shift (which is proportional to cellular dry mass) and formation of big ’blebs’. This explanation is supported by the fluorescent images in Figs. 4(g) and 4(h) and phase contrast image in Fig. 4(i) clearly demonstrating cell membrane rupture and cell lysis.
The dynamics of average phase shift decrease can be used for estimation of the efflux rate of intracellular content. At high irradiation doses the severe membrane damage occurs being partly caused by lipid peroxidation by ROS. The intracellular protective antioxidant mechanisms are unable to recover the damage and cell dies through a fast unprogrammed mechanism, necrosis. The higher is the dose the faster happens the cell membrane rupture and the more rapid is the efflux of intracellular content. The resulting loss of dry mass however is almost the same for the specific type of cells.
Yellow curves in Figs. 2(a) and 2(b) obtained at intermediate irradiation doses and showing a postponed decrease of average phase shift and loss of cellular dry mass with a pre-necrotic phase during first 20-40 minutes, may be interpreted as secondary necrosis [34].
The designated scenarios of cells’ response to photodynamic treatment were found to be similar for the two types of cancer cells used in our experiments. However the irradiation doses related to the specific tendencies described above were substantially different for these cell types (see Fig. 2(c)). A549 cells were found to be more resistant to photodynamic treatment, with all the above processes occurring at significantly higher doses than in HeLa cells.
Note that according to our observations, the level of intracellular accumulation of Radachlorin PS was approximately the same for both the cell lines. Similar results were demonstrated earlier for HeLa and mesenchimal endometrial stem cell lines [20]. Thus, the significant difference in effective irradiation doses can hardly be explained by the level of PS accumulation. However, although both the carcinoma lines overexpress receptors for epidermal growth factor, which in general has proliferative and antiapoptotic effect [35], A549 cells were shown to possess the activated mutant Ki-Ras thus providing additional resistance to apoptotic death [36,37]. Therefore A549 cells may have higher anti-stress potential as compared to HeLa cells, and relatively high doses of ROS were necessary to stimulate stress response in A549 cells.
It should be also emphasized that in this work the very early response to short-term irradiation of PS-loaded cells was monitored. The response manifested typical features of apoptotic and necrotic behavior during first 90-120 min after irradiation in both cell lines in dose-dependent manner. It is well established now that signaling pathways involved in stress response have complicated patterns with numerous feedbacks and the final outcome depends on the cell type. Such delayed consequences may result in cell death by apoptosis or necrosis [38], as well as in cell survival by stimulating autophagy or senescence [39], with the two latter pathways developing over a much longer period of several hours and days. The overall goal of anticancer therapy is to destroy malignant cells without affecting normal ones, in particular the stem cells providing post-treatment regenerative potential. It was also shown, that mesenchymal and embryonic stem cells respond quite differently to elevated levels of ROS and the effects of short- and long-term treatment with H$_2$0$_2$ provide different outcomes [40]. However, early cell responses to the increase in ROS level require additional studies. Non-invasive digital holographic approach for analysis of early changes in the cells under PD treatment is promising for evaluating these effects and their comparison with early stages of stress response in live cells.
5. Conclusions
We have performed a thorough monitoring of cells behavior under photodynamic treatment in vitro at irradiation doses varied in a wide range. Three major pathways of cell death were considered for the two cultured cancer cell lines. Main scenarios of alterations of optical parameters of cells were identified and analyzed. The conclusion made on the changes of cellular parameters in response to treatment was based on the statistical assay with the level of significance p$<$5$\%$. The results obtained demonstrate that different malignancies can be differently responsive to photodynamic treatment. The desired pathway of cell death requires specific treatment parameters depending upon the cell type and applied photosensitizer.
The results obtained suggest that average phase shift of the transmitted wavefront can be used for quantitative non-invasive cell death analysis. This conclusion was confirmed by comparison with fluorescent analysis with standard test assays made on the basis of confocal microscopy.
Funding
Russian Science Foundation (19-14-00108); Council on grants of the President of the Russian Federation for Support of Young Scientists (SP-2349.2019.4).
Disclosures
The authors declare that there are no conflicts of interest related to this article.
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