Fluorescence Microscopy Methods Research Articles

Collagen hydrogel protects intestinal epithelial cells from indomethacin-induced damage: results of an in vitro experiment

INTRODUCTION. Indomethacin is a derivative of indoleacetic acid and has anti-inflammatory, analgesic and antipyretic effects. However, the results of numerous studies show that indomethacin, like many other nonsteroidal anti-inflammatory drugs (NSAIDs), have an inhibitory effect on the viability and functional activity of enterocytes. In this regard, the search for new ways to reduce the severity of side effects from the use of NSAIDs remains relevant. One of these approaches may be to enrich patients’ diets with non-drug biologically active compounds, including proteins. However, the effect of dietary proteins and biologically active peptides on NSAID-induced damage to the wall of the small intestine and stomach has not been sufficiently studied. AIM. To evaluate the ability of a collagen-containing dietary supplement to protect human duodenal epithelial cells (HuTu-80 line) from indomethacin-induced damage. MATERIALS AND METHODS. The composite collagen-containing hydrogel was provided by «FIRST ALIVE COLLAGEN» LLC (Russia) and is a registered dietary supplement. The work used a commercial culture of human skin fibroblast cells and human duodenal epithelial cells (line HuTu-80). The viability of intestinal cells and fibroblasts was assessed using light and fluorescence microscopy and flow cytometry methods. RESULTS AND DISCUSSION. It has been established that indomethacin inhibits cell growth, causes apoptosis and death of enterocytes, and also leads to the accumulation of cells in the S-phase, which indicates a disruption in the regulation of the cell cycle. It was revealed that collagen hydrogel prevents cell death caused by indomethacin and reduces the number of apoptotic cells in the population. The protective effect of collagen hydrogel is characterized by normalization of the cell cycle of enterocytes and restoration of their growth and proliferative activity. CONCLUSION. Thus, collagen hydrogel, in vitro, is able to reduce the pathogenic effect of indomethacin on human intestinal epithelial cells. The protective effect of collagen hydrogel is characterized by maintaining viability, inhibiting apoptotic processes, and maintaining cell cycle stability. The results obtained indicate the prospects of using a dietary supplement based on a composite collagen hydrogel as a prophylactic agent to reduce the risk of NSAID-associated gastrointestinal diseases. However, to confirm the therapeutic effectiveness of the dietary supplement, further research is necessary, both using experimental animal modeling of NSAID-associated diseases of the human gastrointestinal tract, and clinical studies.

A statistical resolution measure of fluorescence microscopy with finite photons

First discovered by Ernest Abbe in 1873, the resolution limit of a far-field microscope is considered determined by the numerical aperture and wavelength of light, approximately λ2NA\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\frac{\\lambda }{2{NA}}$$\\end{document}. With the advent of modern fluorescence microscopy and nanoscopy methods over the last century, this definition is insufficient to fully describe a microscope’s resolving power. To determine the practical resolution limit of a fluorescence microscope, photon noise remains one essential factor yet to be incorporated in a statistics-based theoretical framework. We proposed an information density measure quantifying the theoretical resolving power of a fluorescence microscope in the condition of finite photons. The developed approach not only allows us to quantify the practical resolution limit of various fluorescence and super-resolution microscopy modalities but also offers the potential to predict the achievable resolution of a microscopy design under different photon levels.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

A novel variational approach for multiphoton microscopy image restoration: from PSF estimation to 3D deconvolution

In multi-photon microscopy (MPM), a recent in-vivo fluorescence microscopy system, the task of image restoration can be decomposed into two interlinked inverse problems: firstly, the characterization of the point spread function (PSF) and subsequently, the deconvolution (i.e. deblurring) to remove the PSF effect, and reduce noise. The acquired MPM image quality is critically affected by PSF blurring and intense noise. The PSF in MPM is highly spread in 3D and is not well characterized, presenting high variability with respect to the observed objects. This makes the restoration of MPM images challenging. Common PSF estimation methods in fluorescence microscopy, including MPM, involve capturing images of sub-resolution beads, followed by quantifying the resulting ellipsoidal 3D spot. In this work, we revisit this approach, coping with its inherent limitations in terms of accuracy and practicality. We estimate the PSF from the observation of relatively large beads (approximately 1 μm in diameter). This goes through the formulation and resolution of an original non-convex minimization problem, for which we propose a proximal alternating method along with convergence guarantees. Following the PSF estimation step, we then introduce an innovative strategy to deal with the high level multiplicative noise degrading the acquisitions. We rely on a heteroscedastic noise model for which we estimate the parameters. We then solve a constrained optimization problem to restore the image, accounting for the estimated PSF and noise, while allowing a minimal hyper-parameter tuning. Theoretical guarantees are given for the restoration algorithm. These algorithmic contributions lead to an end-to-end pipeline for 3D image restoration in MPM, that we share as a publicly available Python software. We demonstrate its effectiveness through several experiments on both simulated and real data.

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.

Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan.

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC-MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC-MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans.

Large-field and fast super-resolution microscopic imaging method based on laser interferometry

In recent years, structured illumination microscopy (SIM) has been drawing great attention for both technique development and application. However, conventional SIM, which uses a spatial light modulator (SLM) for fringe projection, often has a limited field of view. To meet the demand for high-throughput microscopic imaging in biomedicine research, a large-field super-resolution (SR) fluorescence microscopic imaging method based on laser interferometry was proposed. The method that combines a two-dimensional (2D) grating for fringe pattern projection and an SLM for selecting fringe orientation can break the limitation of fringe number limited by the digital projection devices. A spatial-domain reconstruction algorithm was developed to improve the computational speed of super-resolution imaging. Finally, an experimental platform for SIM microscopy was established. A large-field view of 1380 μm × 1035 μm under a 20×/NA0.75 objective is experimentally demonstrated, and an enhancement of 1.8-fold resolution is realized. The spatial-domain reconstruction algorithm can significantly improve the computational speed by approximately 10 times faster compared to the traditional frequency-domain algorithm.

The intracellular visualization of exogenous DNA in fluorescence microscopy.

In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.

Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 75 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Effect of Syzygium cumini on Oxidative Stress Induced Cardiac Cellular Anomalies.

Doxorubicin (Dox), an antineoplastic agent is used as a primary anticancerous drug against various types of cancers. However, its associated toxicity to the cardiovascular system is major. Literature has recorded the cases of mortality due to poor validation and lack of prediagnosis of Dox-induced cardiotoxicity. Therapeutic interventions using natural products having cardioprotective properties with low toxic outcomes hold therapeutic potential for future cardio-oncological therapies. Syzygium cumini (Black berry), a traditional Indian herbal plant, has been researched and found to exert cardioprotective, anti-inflammatory, and antioxidant activities, which have been credited due to the presence of polyphenols, flavonoids, and tannins. In the current research, we investigated the cardioprotective potential of Syzygium cumini against Doxorubicin-induced cardiotoxicity (DIC) in H9C2 cardiomyocytes. Methanolic seed extract preparation of Syzygium cumini was performed using the Soxhlet apparatus. Cell viability and cell death assays were performed to determine the cardiotoxic doses of Doxorubicin. Furthermore, the cardioprotective potential of Syzygium cumini extract against DIC was studied. Morphological and nuclear alterations in H9C2 cells were studied by microscopic assays using Giemsa, Haematoxylin-Eosin stain, and PI. The intracellular stress level and ROS production were studied using DCFH-DA followed by mitochondrial integrity analysis using fluorescent microscopic methods. In the results, we investigated that Dox exerted a dose and time-dependent cardiotoxicity on H9C2 cardiomyocytes. Moreover, we observed that morphological and nuclear alterations caused by doxorubicin in dose-dependent manner were prevented by supplementing with Syzygium cumini polyphenols and it attenuated the oxidative stress in H9C2 cardiomyocytes effectively. Conclusively, Syzygium cumini possesses cardioprotective potential in H9C2 cardiomyocytes in dox-induced cardiotoxicity.

Optically Imaging In Situ Effects of Electrochemical Cycling on Single Nanoparticle Electrocatalysis.

Single-nanoparticle studies often need one or a series of nanoparticle populations that are designed with differences in a nominally particular structural parameter to clarify the structure-activity relationship (SAR). However, the heterogeneity of various properties within any population would make it rather difficult to approach an ideal one-parameter control. In situ modification ensures the same nanoparticle to be investigated and also avoids complicating effects from the otherwise often needed ex situ operations. Herein, we apply electrochemical cycling to single platinum nanoparticles and optically examine their SAR. An electrocatalytic fluorescent microscopic method is established to evaluate the apparent catalytic activity of a number of single nanoparticles toward the oxygen reduction reaction. Meanwhile, dark-field microscopy with the substrate electrode under a cyclic potential control is found to be able to assess the electrochemically active surface area (ECSA) of single nanoparticles via induced chloride redox electrochemistry. Consequently, nanoparticles with drastically increased catalytic activity are discovered to have larger ECSAs upon potential regulation, and interestingly, there are also a few particles with decreased activity, as opposed to the overall trend, that all develop a smaller ECSA in the process. The deactivated nanoparticles against the overall enhancement effects of potential cycling are revealed for the first time. As such, the SAR of single nanoparticles when subjected to an in situ structural control is optically demonstrated.

An Indirect Fluorescence Microscopy Method to Assess Vaginal Lactobacillus Concentrations.

Lactobacillus species are the main colonizers of the vaginal microbiota in healthy women. Their absolute quantification by culture-based methods is limited due to their fastidious growth. Flow cytometry can quantify the bacterial concentration of these bacteria but requires the acquisition of expensive equipment. More affordable non-culturable methods, such as fluorescence microscopy, are hampered by the small size of the bacteria. Herein, we developed an indirect fluorescence microscopy method to determine vaginal lactobacilli concentration by determining the correlation between surface area bacterial measurement and initial concentration of an easily cultivable bacterium (Escherichia coli) and applying it to lactobacilli fluorescence microscopy counts. In addition, vaginal lactobacilli were quantified by colony-forming units and flow cytometry in order to compare these results with the indirect method results. The colony-forming-unit values were lower than the results obtained from the other two techniques, while flow cytometry and fluorescence microscopy results agreed. Thus, our developed method was able to accurately quantify vaginal lactobacilli.

Detection of CPSF6 in Biomolecular Condensates as a Reporter of HIV-1 Nuclear Import.

The initial stages of HIV-1 infection involve the transport of the viral core into the nuclear compartment. The presence of the HIV-1 core in the nucleus triggers the translocation of CPSF6/CPSF5 from paraspeckles into nuclear speckles, forming puncta-like structures. While this phenomenon is well-documented, the efficiency of CPSF6 translocation to nuclear speckles upon HIV-1 infection varies depending on the type of cell used. In some human cell lines, only 1-2% of the cells translocate CPSF6 to nuclear speckles when exposed to a 95% infection rate. To address the issue that only 1-2% of cells translocate CPSF6 to nuclear speckles when a 95% infection rate is achieved, we screened several human cell lines and identified a human a cell line in which approximately 85% of the cells translocate CPSF6 to nuclear speckles when 95% infection rate is achieved. This cellular system has enabled the development of a robust fluorescence microscopy method to quantify the translocation of CPSF6 into nuclear speckles following HIV-1 infection. This assay holds the potential to support studies aimed at understanding the role of CPSF6 translocation to nuclear speckles in HIV-1 infection. Additionally, since the translocation of CPSF6 into nuclear speckles depends on the physical presence of the viral core in the nucleus, our method also serves as a reporter of HIV-1 nuclear import.

DNase improves the efficacy of antimicrobial photodynamic therapy in the treatment of candidiasis induced with Candida albicans.

The study evaluated the association of DNase I enzyme with antimicrobial photodynamic therapy (aPDT) in the treatment of oral candidiasis in mice infected with fluconazole-susceptible (CaS) and -resistant (CaR) Candida albicans strains. Mice were inoculated with C. albicans, and after the infection had been established, the tongues were exposed to DNase for 5 min, followed by photosensitizer [Photodithazine®(PDZ)] and light (LED), either singly or combined. The treatments were performed for 5 consecutive days. Treatment efficacy was evaluated by assessing the tongues via fungal viable population, clinical evaluation, histopathological and fluorescence microscopy methods immediately after finishing treatments, and 7 days of follow-up. The combination of DNase with PDZ-aPDT reduced the fungal viability in mice tongues immediately after the treatments by around 4.26 and 2.89 log10 for CaS and CaR, respectively (versus animals only inoculated). In the fluorescence microscopy, the polysaccharides produced by C. albicans and fungal cells were less labeled in animals treated with the combination of DNase with PDZ-aPDT, similar to the healthy animals. After 7 days of the treatment, DNase associated with PDZ-aPDT maintained a lower count, but not as pronounced as immediately after the intervention. For both strains, mice treated with the combination of DNase with PDZ-aPDT showed remission of oral lesions and mild inflammatory infiltrate in both periods assessed, while animals treated only with PDZ-aPDT presented partial remission of oral lesions. DNase I enzyme improved the efficacy of photodynamic treatment.

Expression of human papillomavirus genotype 52 pseudovirus in HEK-293T cells

Human papillomavirus (HPV) is the predominant cause of cervical cancer globally. HPV genotype 52 is categorized as a high-risk type and holds the highest prevalence in Indonesia (23.2 %). The HPV pseudovirus can be used to test the immunogenicity of HPV vaccine candidates and evaluate the neutralization efficiency of the antibodies against the virus. The aim of this study was to express the HPV52 L1 and L2 capsid protein into pseudovirion containing the GFP reporter plasmid (pfwB) in HEK-293T cells. HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfwB were amplified in E. coli. The plasmids were extracted and co-transfected using polyethyleneimine (PEI)-based transfection method into the HEK-293T cells. The expression of HPV52 pseudoviruses were confirmed by fluorescence microscopy and western blot method. Co-transfection of HPV52 L1 and L2 coding plasmid (pVITRO52) along with pfWB into the HEK-293T cells was successfully carried out. The co–transfected HEK-293T cells showed fluorescence. The western blot assay using HPV52 L1 protein primary antibody showed a band around ∼55 kDa. In the future, the results of this study will be used to evaluate the immunogenicity and neutralization assay of HPV vaccine candidates.

Preparation of mcl-polyhydroxyalkanoate nanoparticles stabilized by the Tween 80 nonionic surfactant

Nanoparticles based on biodegradable polymers find numerous applications in medicine as substances for intracellular drug delivery. Biosynthetically produced poly-3-hydroxyalkanoates (P3HAs) are among the most promising polymers of a lipid nature. In particular, polyhydroxybutyrate and polyhydroxyvalerate (scl-poly-3-hydroxyalkanoates) are widely used compounds, which are soluble only in organochlorine solvents. The use of organochlorine solvents faces several obstacles, since such chemicals may exhibit carcinogenic effects on the human body. However, the P3Has compounds consisting of hydroxybutyric acid residues with 6–14 carbon atoms in the main chain (mcl-poly-3-hydroxyalkanoates) are soluble not only in CHCl3 or CH2Cl2 but also in paraffins. These hydrocarbons, such as n-hexane, can be easily separated from aqueous solutions and are not known to exhibit high cytotoxicity. Consequently, the application of mcl-poly-3-hydroxyalkanoates can prevent the contamination of prepared dosage forms with organochlorine compounds. To this end, a methodology for the synthesis of mcl-P3HA nanoparticles stabilized with the Tween 80 nonionic surfactant was proposed. The ratio between the concentration of P3HA and the detergent was optimized. The present study revealed that the obtained particles have an average size of 200±90 nm and a zeta potential of -17±5 mV. Upon investigating the stability of the particle solution at 4 °C, it was found that the nanoparticles did not alter their size and zeta potential for 90 days. The fluorescence microscopy method showed that they could be delivered into BHK-21 cells within 2 h. In addition, the nanoparticles synthesized had no toxic effect on BHK-21 cells at a concentration of up to 200 μg/mL.

EFFECT OF USING MULTIPOTENT MESENCHYMAL STROMAL CELLS IN BONE GRAFTING WITH XENOGENIC BIOMATERIALS

Currently, there is a significant frequency of degenerative diseases of the spine after surgical interventions with the use of bone grafting, especially in elderly patients. The results of the research indicate that the use of stem cells is one of the promising areas to increase the efficiency of osseointegration. The aim of the study was to evaluate the effectiveness of osteogenesis in the conditions of using xenografts loaded with stem cells, as well as morphological features of osseointegration. Materials and methods. An experimental study was conducted on 22 male rabbits. 2 animals were used to obtain stem cells, the remaining animals were implanted with xenogenic bone–substituting material "Osteomatrix" in the iliac wing, of which 10 animals were implanted with a xenograft not populated with cells, and the other 10 animals were implanted with an identical xenograft populated with mesenchymal stromal cells. Withdrawal from the experiment was carried out on the 60th day after implantation. The material's examination was carried out using the methods of fluorescence and light microscopy. To assess the statistical significance of observed differences (evaluation of proliferation, neoangiogenesis and osseointegration) in the experimental and control groups, the Mann–Whitney U test was used with a critical significance of p ≤ 0.05. Results. When using an osteoplastic material that was not loaded with multipotent mesenchymal stromal cells within 60 days after surgery, osseointegration passes through indirect osteogenesis with formation of full-fledged bone tissue in the future, which increases the time of complete defect healing. In the case of using an osteomatrix loaded with multipotent mesenchymal stromal cells of the bone marrow, osteogenesis proceeds in a direct way with the formation of a full-fledged bone tissue. Osteomatrix loading with multipotent mesenchymal stromal cells stimulates neoangiogenesis and proliferative activity of the tissue, which promotes activation of bone tissue repair processes and stimulates the processes of xenograft osseointegration. Conclusions. The use of multipotent mesenchymal stromal cells in bone grafting using xenografts increases the efficiency of osseointegration by stimulating direct osteogenesis, increasing the activity of proliferation and angiogenesis.

Cerium Oxide Nanoparticles Protect Cortical Astrocytes from Oxygen-Glucose Deprivation through Activation of the Ca2+ Signaling System.

Most of the works aimed at studying the cytoprotective properties of nanocerium are usually focused on the mechanisms of regulation of the redox status in cells while the complex effects of nanocerium on calcium homeostasis, the expression of pro-apoptotic and protective proteins are generally overlooked. There is a problem of a strong dependence of the effects of cerium oxide nanoparticles on their size, method of preparation and origin, which significantly limits their use in medicine. In this study, using the methods of molecular biology, immunocytochemistry, fluorescence microscopy and inhibitory analysis, the cytoprotective effect of cerium oxide nanoparticles obtained by laser ablation on cultured astrocytes of the cerebral cortex under oxygen-glucose deprivation (OGD) and reoxygenation (ischemia-like conditions) are shown. The concentration effects of cerium oxide nanoparticles on ROS production by astrocytes in an acute experiment and the effects of cell pre-incubation with nanocerium on ROS production under OGD conditions were studied. The dose dependence for nanocerium protection of cortical astrocytes from a global increase in calcium ions during oxygen-glucose deprivation and cell death were demonstrated. The concentration range of cerium oxide nanoparticles at which they have a pro-oxidant effect on cells has been identified. The effect of nanocerium concentrations on astrocyte preconditioning, accompanied by increased expression of protective proteins and limited ROS production induced by oxygen-glucose deprivation, has been investigated. In particular, a correlation was found between an increase in the concentration of cytosolic calcium under the action of nanocerium and the suppression of cell death. As a result, the positive and negative effects of nanocerium under oxygen-glucose deprivation and reoxygenation in astrocytes were revealed at the molecular level. Nanocerium was found to act as a "double-edged sword" and to have a strictly defined concentration therapeutic "window".

Super-Resolution Imaging of Neuronal Structures with Structured Illumination Microscopy.

Super-resolution structured illumination microscopy (SR-SIM) is an optical fluorescence microscopy method which is suitable for imaging a wide variety of cells and tissues in biological and biomedical research. Typically, SIM methods use high spatial frequency illumination patterns generated by laser interference. This approach provides high resolution but is limited to thin samples such as cultured cells. Using a different strategy for processing raw data and coarser illumination patterns, we imaged through a 150-micrometer-thick coronal section of a mouse brain expressing GFP in a subset of neurons. The resolution reached 144 nm, an improvement of 1.7-fold beyond conventional widefield imaging.

Resolution enhancement with a task-assisted GAN to guide optical nanoscopy image analysis and acquisition

Super-resolution fluorescence microscopy methods enable the characterization of nanostructures in living and fixed biological tissues. However, they require the adjustment of multiple imaging parameters while attempting to satisfy conflicting objectives, such as maximizing spatial and temporal resolution while minimizing light exposure. To overcome the limitations imposed by these trade-offs, post-acquisition algorithmic approaches have been proposed for resolution enhancement and image-quality improvement. Here we introduce the task-assisted generative adversarial network (TA-GAN), which incorporates an auxiliary task (for example, segmentation, localization) closely related to the observed biological nanostructure characterization. We evaluate how the TA-GAN improves generative accuracy over unassisted methods, using images acquired with different modalities such as confocal, bright-field, stimulated emission depletion and structured illumination microscopy. The TA-GAN is incorporated directly into the acquisition pipeline of the microscope to predict the nanometric content of the field of view without requiring the acquisition of a super-resolved image. This information is used to automatically select the imaging modality and regions of interest, optimizing the acquisition sequence by reducing light exposure. Data-driven microscopy methods like the TA-GAN will enable the observation of dynamic molecular processes with spatial and temporal resolutions that surpass the limits currently imposed by the trade-offs constraining super-resolution microscopy.