Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for visualizing the distribution of fluorescence lifetimes of fluorophores within a tissue, and is used for optical metabolic imaging. However, processing the large volumes of data generated by FLIM can be time-consuming and challenging for an untrained operator. Convolutional neural networks (CNNs) have demonstrated its effectiveness for tissue image and FLIM segmentation, making them a promising tool for automated analysis of FLIM images. The article presents a method for segmenting FLIM images of liver tissue during the regeneration using a CNN based on the UNet++ architecture. The CNN was trained to detect cell boundaries and nuclei, and the resulting masks were used to approximate the fluorescence decay curves for the FLIM-image data. The results show that the CNN is effective in segmenting FLIM images of cells and tissues, and obtaining fluorescence decay curves. The use of CNNs and other machine learning methods in medical image segmentation and analysis will improve and speed up diagnosis and reduce diagnostic errors.

BACKGROUND: Methods for microbial inactivation, including physical approaches aimed at the destruction of biofilms formed by uropathogenic microorganisms for the prevention of infectious and inflammatory diseases in urology, remain insufficiently studied. The development of new strategies in this field remains relevant. AIM: To evaluate the feasibility of photodynamic inactivation of biofilms formed by typical representatives of uropathogenic microorganisms using an antiseptic agent with a bacteriostatic effect—methylene blue—possessing photochemical properties. METHODS: Cultures of Staphylococcus aureus and Escherichia coli isolated from renal calculi of patients from a urology department were used. In vitro experiments on photodynamic inactivation of microorganisms were conducted on mature preformed biofilms. Irradiation was performed using a diode laser emitting at a wavelength of 662 nm through a sterile 0.1% methylene blue solution in continuous mode across five setups (three control, two experimental). After irradiation, biofilms on the cover glasses were fixed on microscope slides using colorless varnish. The prepared specimens were stained with acridine orange solution, dried in the dark, examined under a fluorescence microscope at ×100 magnification using an immersion system, and photographed with a digital camera. Images were digitally processed using 3D modeling technologies with ImageJ software version 1.52a. RESULTS: The impact of the photoactive agent and laser irradiation was assessed at two power settings—450 mW and 1100 mW. In the first case, partial destruction of the biofilms was noted (41.9% of the original biofilm structure for S. aureus and 82.4% for E. coli), whereas in the second case, exposure at 1100 mW resulted in complete degradation of the mature multilayer biofilm into single cells without extracellular matrix, corresponding to 97.7% destruction of the original biofilm structure for S. aureus and 96.5% for E. coli. CONCLUSION: This study is the first to demonstrate the feasibility of photodynamic inactivation of uropathogenic biofilm-forming microorganisms using a photochemically active agent—methylene blue. The promising results suggest that combined laser irradiation and methylene blue application may serve as an alternative or adjunct to systemic antibiotic therapy in urological practice.

The most effective method of treating tumors localized in the liver remains resection. However, in the presence of concomitant pathology, the regenerative potential of the liver is significantly reduced. To date, there is insufficient fundamental data on the mechanisms responsible for the disruption of liver regeneration, and there is no effective method for assessing its regenerative potential. The most suitable model for these purposes is acute liver injury (ALI). Modern non-contrast methods of multiphoton microscopy with second harmonic generation and fluorescence lifetime imaging microscopy (FLIM) modes enable intravital evaluation of the metabolic status of the hepatocytes; therefore, this expands the possibilities for studying the processes occurring in cells during regeneration in the context of any pathologies.

In this paper, we report on a study regarding the efficiency of the post-operational phototherapy of the tumor bed after resection with both a cold knife and a laser scalpel in laboratory mice with CT-26 tumors. Post-operational processing included photodynamic therapy (PDT) with a topically applied chlorin-based photosensitizer (PS), performed at wavelengths of 405 or 660 nm, with a total dose of 150 J/cm2. The selected design of the tumor model yielded zero recurrence in the laser scalpel group and 92% recurrence in the cold knife group without post-processing, confirming the efficiency of the laser scalpel in oncology against the cold knife. The application of PDT after the cold knife resection decreased the recurrence rate to 70% and 42% for the 405 nm and 660 nm procedures, respectively. On the other hand, the application of PDT after the laser scalpel resection induced recurrence rates of 18% and 30%, respectively, for the considered PDT performance wavelengths. The control of the penetration of PS into the tumor bed by fluorescence confocal microscopy indicated the deeper penetration of PS in the case of the cold knife, which presumably provided deeper PDT action, while the low-dose light exposure of deeper tissues without PS, presumably, stimulated tumor recurrence, which was also confirmed by the differences in the recurrence rate in the 405 and 660 nm groups. Irradiation-only light exposures, in all cases, demonstrated higher recurrence rates compared to the corresponding PDT cases. Thus, the PDT processing of the tumor bed after resection could only be recommended for the cold knife treatment and not for the laser scalpel resection, where it could induce tumor recurrence.

Photodynamic therapy (PDT) is a minimally invasive method for cancer treatment, one of the effects of which is the oxidation of membrane lipids. However, changes in biophysical properties of lipid membranes during PDT have been poorly explored. In this work, we investigated the effects of PDT on membrane microviscosity in cancer cells in the culture and tumor xenografts. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive rotor BODIPY2. It was found that PDT using chlorine e6-based photosensitizer Photoditazine caused a quick, steady elevation of membrane microviscosity both in cellulo and in vivo. The proposed mechanisms responsible for the increase in microviscosity was lipid peroxidation by reactive oxygen species that resulted in a decrease of phosphatidylcholine and the fraction of unsaturated fatty acids in the membranes. Our results suggest that the increased microviscosity is an important factor that contributes to tumor cell damage during PDT.

Background: Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood. Methods: In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor. Results: Depending on the localization of the phototoxic protein, different effects on membrane microviscosity were observed. With nuclear localization of KillerRed, a gradual decrease in microviscosity was detected throughout the entire observation period, while for membrane localization of KillerRed, a dramatic increase in microviscosity was observed in the first minutes after PDT, and then a significant decrease at later stages of monitoring. The obtained data on cell monolayers are in good agreement with the data obtained for 3D tumor spheroids. Conclusions: These results indicate the involvement of membrane microviscosity in the response of tumor cells to PDT, which strongly depends on the localization of reactive oxygen species attack via targeting of a genetically encoded photosensitizer.

To reduce the risk of post-hepatectomy liver failure in patients with hepatic pathologies, it is necessary to develop an approach to express the intraoperative assessment of the liver's regenerative potential. Traditional clinical methods do not enable the prediction of the function of the liver remnant. Modern label-free bioimaging, using multiphoton microscopy in combination with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), can both expand the possibilities for diagnosing liver pathologies and for assessing the regenerative potential of the liver. Using multiphoton and SHG microscopy, we assessed the structural state of liver tissue at different stages of induced steatosis and fibrosis before and after 70% partial hepatectomy in rats. Using FLIM, we also performed a detailed analysis of the metabolic state of the hepatocytes. We were able to determine criteria that can reveal a lack of regenerative potential in violated liver, such as the presence of zones with reduced NAD(P)H autofluorescence signals. Furthermore, for a liver with pathology, there was an absence of the jump in the fluorescence lifetime contributions of the bound form of NADH and NADPH the 3rd day after hepatectomy that is characteristic of normal liver regeneration. Such results are associated with decreased intensity of oxidative phosphorylation and of biosynthetic processes in pathological liver, which is the reason for the impaired liver recovery. This modern approach offers an effective tool that can be successfully translated into the clinic for express, intraoperative assessment of the regenerative potential of the pathological liver of a patient.

A decrease in the regenerative potential of the liver during the development of non-alcoholic fatty liver disease (NAFLD), which is observed in the vast majority of patients with diabetes mellitus type 1, significantly increases the risk of postoperative liver failure. In this regard, it is necessary to develop new approaches for the rapid intraoperative assessment of the condition of liver tissue in the presence of concomitant liver pathology. A modern label-free approach based on multiphoton microscopy, second harmonic generation (SHG), and fluorescence lifetime imaging microscopy (FLIM) allow for the evaluation of the structure of liver tissue as well as the assessment of the metabolic state of hepatocytes, even at the cellular level. We obtained optical criteria and identified specific changes in the metabolic state of hepatocytes for a reduced liver regenerative potential in the presence of induced diabetes mellitus type 1. The obtained criteria will expand the possibilities for the express assessment of the structural and functional state of liver tissue in clinical practice.

In the recent years promising results have been shown by the use of Compression optical coherence elastography (C-OCE) as a new optical biopsy approach to morphological assessment/diagnostics of human breast cancer using differences in elastic properties of morphological components of cancerous tissues. In this study, for the first time, a relationship was established between microstructural organization and biomechanical properties of breast-cancer tissue with pathomorphological changes caused by chemotherapy. To characterize texture of collagen fibers in the microenvironment of breast cancer, high-resolution visualization by Second-harmonic generation (SHG) microscopy was used. A side-by-side C-OCE and SHG imaging of patients' breast cancer tissues before and after chemotherapy was carried out. Regions of the cancer stroma (collagen fibers outside aggregates of cancer cells) were assessed separately from regions of cancer cell clusters penetrated by collagen fibers. For cancer stroma areas after chemotherapy, a statistically significant decrease in stiffness values was found. Simultaneously, parameters of collagen texture in SHG images (mean intensity, "coherency" and "energy") indicated increase in the collagen content, orientational orderliness, and collagen-texture heterogeneity. In contrast, cancer-cell areas post chemotherapy showed a statistically significant increase in stiffness. Analysis of SHG images of these regions indicated decrease in the inter-cellular collagen content and heterogeneity of its texture, whereas its orientational orderliness somewhat increased. The established negative correlation between stiffness and SHG parameters of collagen in cancer stroma indicates the contribution of the increase in orientational orderliness and total collagen content to the reduction in stiffness of breast cancer stroma after chemotherapy. For cancer-cell regions, significantly lower correlation between stiffness and SHG parameters (especially for coherency) was found, indicating stronger role of chemotherapy-induced changes in cancer cells themselves. These results give a deeper insight in the role of collagen texture organization in biomechanics of breast cancer tissues and contribute to a more detailed substantiation of the morphological characterization of breast cancer by C-OCE imaging.

Antimicrobial photodynamic therapy (aPDT) was demonstrated to be effective against various species of Gram-positive bacteria. However, the complex structure of a Gram-negative bacteria envelope limits the application of aPDT. Thus, the goal of this study was to improve the efficiency of antimicrobial photodynamic therapy with Fotoditazin against uropathogenic Gram-negative bacteria. The non-ionic detergent Triton X-100 and emulsifier Tween 80 were tested. The effect of extracellular photosensitizer on aPDT efficacy was analyzed. Moreover, the irradiation regime was optimized in terms of the output power and emitting mode. It was found that Triton X-100 at 10% vol enhanced the efficacy of aPDT of E. coli up to 52%. The subsequent observation demonstrated that, when the photosensitizer was removed from the extracellular space, the efficacy of aPDT on various Gram-negative species decreased dramatically. As for the irradiation mode, an increase in the laser output power led to an increase in the aPDT efficacy. The pulsed irradiation mode did not affect the aPDT efficacy. Thus, in order to achieve optimal aPDT efficacy, bacteria should be irradiated at 450-mW output power in the presence of Triton X-100 and a photosensitizer in the extracellular environment. However, it should be noted that the efficacy of aPDT of K. pneumoniae was significantly lower than for other species. The developed aPDT technique may be effective in a native environment of uropathogenic microorganisms.

Urinary tract infection (UTIs) aremainly caused by a number of anatomical and physiological dysfunctions, but there are also some iatrogenic factors, including the use of certain medications, that contribute to the development of UTIs. The virulence of bacteria that colonize the urinary tract may be modified by pH and by the presence of soluble substances in urine, such as norepinephrine (NE) and glucose. In this work, we studied the influence of NE and glucose across a range of pHs (5, 7, 8) on the biomass, matrix production and metabolism of uropathogenic strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus faecalis. We used Congo red and gentian violet to stain the extracellular matrix and biomass, respectively, of biofilms. The optical density of staining of the biofilms was measured using a multichannel spectrophotometer. The metabolic activity was analyzed by MTT assay. It was shown that NE and glucose stimulate biomass production both in the Gram-negative and Gram-positive uropathogens. The metabolic activity in the presence of glucose was higher at pH 5 for E. coli (in 4.0 ± 0.1 times), Ps. aeruginosa (in 8.2 ± 0.2 times) and Kl. pneumoniae (in 4.1 ± 0.2 times). Matrix production of Kl. pneumoniae increased under NE (in 8.2 ± 0.2 times) and in the presence of glucose (in 1.5 ± 0.3 times). Thus, NE and glucose in urine may lead to persistent UTI under patient stress and in the case of metabolic glucose disorders.

Abuse with hepatotoxic agents is a major cause of acute liver failure. The search for new criteria indicating the acute or chronic pathological processes is still a challenging issue that requires the selection of effective tools and research models. Multiphoton microscopy with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM) are modern label-free methods of optical biomedical imaging for assessing the metabolic state of hepatocytes, therefore reflecting the functional state of the liver tissue. The aim of this work was to identify characteristic changes in the metabolic state of hepatocytes in precision-cut liver slices (PCLSs) under toxic damage by some of the most common toxins: ethanol, carbon tetrachloride (CCl4) and acetaminophen (APAP), commonly known as paracetamol. We have determined characteristic optical criteria for toxic liver damage, and these turn out to be specific for each toxic agent, reflecting the underlying pathological mechanisms of toxicity. The results obtained are consistent with standard methods of molecular and morphological analysis. Thus, our approach, based on optical biomedical imaging, is effective for intravital monitoring of the state of liver tissue in the case of toxic damage or even in cases of acute liver injury.

Liver regeneration has been studied for many decades, and the mechanisms underlying regeneration of normal liver following resection are well described. However, no less relevant is the study of mechanisms that disrupt the process of liver regeneration. First of all, a violation of liver regeneration can occur in the presence of concomitant hepatic pathology, which is a key factor reducing the liver's regenerative potential. Understanding these mechanisms could enable the rational targeting of specific therapies to either reduce the factors inhibiting regeneration or to directly stimulate liver regeneration. This review describes the known mechanisms of normal liver regeneration and factors that reduce its regenerative potential, primarily at the level of hepatocyte metabolism, in the presence of concomitant hepatic pathology. We also briefly discuss promising strategies for stimulating liver regeneration and those concerning methods for assessing the regenerative potential of the liver, especially intraoperatively.

The 'dusting' technique of lithotripsy for the removal of infected urinary calculi and the wide use of drainage after endoscopic surgery may stimulate spreading of multidrug-resistant bacterial strains. Antibacterial photodynamic therapy (PDT) is one promising method for the elimination these strains. The purpose of our study was to evaluate alterations of renal pelvis morphology and renal function in laboratory animals after bactericidal regimens of PDT. Renal pelvises of pigs were filled with Photoditazine and then assessed either by examining the accumulation of Photoditazine in the urothelium or by illumination with a laser at a wavelength of 662 nm. A renal test and a complete blood count was performed to assess a negative effect of the treatment on health. Structural alterations of the kidney tissues were analyzed by histological examination. No photosensitizer fluorescence was detected in the urothelium of the pelvis. Histological study showed that PDT caused minor changes to the urothelium of the renal pelvis but did not affect the underlying connective tissue. No renal function abnormalities were found after PDT. Thus, the study indicates that antibacterial PDT is a safety technique that can complement common antibiotic therapy in the surgical treatment of urolithiasis.

BackgroundThere is an urgent clinical need for targeted strategies aimed at the treatment of bone defects resulting from fractures, infections or tumors. 3D scaffolds represent an alternative to allogeneic MSC transplantation, due to their mimicry of the cell niche and the preservation of tissue structure. The actual structure of the scaffold itself can affect both effective cell adhesion and its osteoinductive properties. Currently, the effects of the structural heterogeneity of scaffolds on the behavior of cells and tissues at the site of damage have not been extensively studied.MethodsBoth homogeneous and heterogeneous scaffolds were generated from poly(L-lactic acid) methacrylated in supercritical carbon dioxide medium and were fabricated by two-photon polymerization. The homogeneous scaffolds consist of three layers of cylinders of the same diameter, whereas the heterogeneous (gradient pore sizes) scaffolds contain the middle layer of cylinders of increased diameter, imitating the native structure of spongy bone. To evaluate the osteoinductive properties of both types of scaffold, we performed in vitro and in vivo experiments. Multiphoton microscopy with fluorescence lifetime imaging microscopy was used for determining the metabolic states of MSCs, as a sensitive marker of cell differentiation. The results obtained from this approach were verified using standard markers of osteogenic differentiation and based on data from morphological analysis.ResultsThe heterogeneous scaffolds showed improved osteoinductive properties, accelerated the metabolic rearrangements associated with osteogenic differentiation, and enhanced the efficiency of bone tissue recovery, thereby providing for both the development of appropriate morphology and mineralization.ConclusionsThe authors suggest that the heterogeneous tissue constructs are a promising tool for the restoration of bone defects. And, furthermore, that our results demonstrate that the use of label-free bioimaging methods can be considered as an effective approach for intravital assessment of the efficiency of differentiation of MSCs on scaffolds.

Using Becker & Hickl TCSPC FLIM systems with fast TCSPC modules and fast detectors, we found ultra-fast fluorescence decay components in biological material, such as mushroom spores, pollen grains, plant tissue, and malignant melanoma. The component decay times are on the order of 10 to 50 ps, the amplitudes range from about 60% to more than 99%. In general, the shortest lifetimes and the largest amplitudes of the fast component were found in strongly coloured material, such as black mushroom spores and melanoma tissue. However, the lifetime not always correlates with the colour. In particular, a fast decay component was not found in tissue from basal cell papilloma, although it is dark brown. This may open a way to identify melanoma tissue and melanoma cells, and thus provide a new tool to investigate melanoma progression.

To develop new methods for the diagnosis and treatment of such a widespread disease as steatosis, there is still a lack of fundamental biological knowledge about various aspects of the functioning of the liver tissue at the cellular level. In our work, we assessed the metabolic state of hepatocytes, as well as the collagen content in the liver tissue with induced steatosis using the modern label-free minimally invasive methods of multiphoton laser scanning microscopy with TPEF mode (Two-Photon Excited Fluorescence) and SHG mode (Second Harmonic Generation), equipped with FLIM (Fluorescence-Lifetime Imaging Microscopy). Using multiphoton microscopy, it was shown that during the development of steatosis, it is possible to identify areas with a reduced NAD(P)H autofluorescence signal in damaged hepatocytes. Using SHG we showed a gradual accumulation of collagen in the liver tissue with induced steatosis, however, extensive areas of fibrosis were not detected even at the advanced stages of the pathology. Using FLIM, we studied the specific features of the energy metabolism of hepatocytes based on data on the lifetimes of various forms of NAD(P)H and their relative contributions. It has been revealed that there is a gradual decrease in the intensity of oxidative phosphorylation, accompanied by the rise in the intensity of lipogenesis in the liver tissue with induced steatosis. Such results are consistent with the data of histological analysis. The results obtained in this work can be useful for developing new criteria for express intraoperative assessment of liver pathology at the cellular level in the clinic.

Lichen sclerosus (LS) is the most common vulvar dermatosis, which is characterized by damage to the connective tissue of the dermis. The affected area in lichen sclerosus is characterized by a sequential change in the main components of the connective tissue - collagen and elastin fibers. The affected area is polymorphic and remains poorly defined from a histological point of view. Among histopathologists, there are no unequivocal opinions on changes in the connective tissue of the dermis in LS. However, an assessment of the degree of dermis damage is important for the timely diagnosis of the condition and adequate treatment. Nonlinear microscopy includes second-harmonic generation (SHG) and two-photon autofluorescence (TPEF). SHG allows to selectively examine the signal from heterotypic collagen fibers of the dermis that contain type 1 collagen. TPEF allows to identify elastic fibers of the connective tissue matrix. It has been demonstrated that nonlinear microscopy allows visualizing the changes in the microstructure of collagen and elastin fibers. Three histological patterns were revealed as a result of the analysis of the nonlinear optical microscopy of the classical VLS. These histological patterns cannot be distinguished using histological stains and indicate a polymorphism of connective tissue changes. Nonlinear microscopy makes it possible to assess the changes in tissue structure, which is important for the histological interpretation of changes in the dermis and to clarify histological classification system in the future.

The degree of virulence correlates with adhesion, biofilm formation, motility and the capacity to quickly colonize biological surfaces. The virulence of the bacteria that have colonized the urinary tract may be modified by substances dissolved in urine. One such substance is the norepinephrine (NE) hormone, which may be present in human urine, especially in times of stress and under changes in the activity of the renin-angiotensin-aldesterone system. In this work, we study the influence of NE on the biomass, biofilm formation, matrix production, adhesion, motility and metabolism of uropathogenic strains of E. coli and S. aureus. We used Congo red and gentian violet staining for detection of matrix and biomass formation, respectively. The optical density was measured by a multichannel spectrophotometer. The motility of bacterial cells was measured on semi-solid agar at 24 h and 48 h. The metabolic activity was analyzed by MTT assay. It was shown that the metabolic activity of E. coli was stimulated by NE, which led to the increasing synthesis of virulence factors such as biofilm production, adhesion, and motility. At the same time, NE did not activate the S. aureus strain’s metabolism and did not change its adhesion and motility. Thus, the virulence activity of uropathogenic E. coli may be modified by NE in urine.