Confocal Microscopy Methods Research Articles

Harnessing Electron Spin Hyperpolarization in Chromophore-Radical Spin Probes for Subcellular Resolution in Electron Paramagnetic Resonance Imaging: Concept and Feasibility.

Obtaining a subcellular resolution for biological samples doped with stable radicals at room temperature (RT) is a long-sought goal in electron paramagnetic resonance imaging (EPRI). The spatial resolution in current EPRI methods is constrained either because of low electron spin polarization at RT or the experimental limitations associated with the field gradients and the radical linewidth. Inspired by the recent demonstration of a large electron spin hyperpolarization in chromophore-nitroxyl spin probe molecules, the present work proposes a novel optically hyperpolarized EPR imaging (OH-EPRI) method, which combines the optical method of two-photon confocal microscopy for hyperpolarization generation and the rapid scan (RS) EPR method for signal detection. An important aspect of OH-EPRI is that it is not limited by the abovementioned restrictions of conventional EPRI since the large hyperpolarization in the spin probes overcomes the poor thermal spin polarization at RT, and the use of two-photon optical excitation of the chromophore naturally generates the required spatial resolution, without the need for any magnetic field gradient. Simulations based on time-dependent Bloch equations, which took into account both the RS field modulation and the hyperpolarization generation by optical means, were performed to examine the feasibility of OH-EPRI. The simulation results revealed that a spatial resolution of up to 2 fL can be achieved in OH-EPRI at RT under in vitro conditions. Notably, the majority of the requirements for an OH-EPRI experiment can be fulfilled by the currently available technologies, thereby paving the way for its easy implementation. Thus, the proposed method could potentially bridge the sensitivity gap between the optical and magnetic imaging techniques.

Direct Visualization of Dynamic Mobility of Li2O2 in Li-O2 Batteries: A Differential Interference Microscopy Study.

The reversibility and the discharge/charge performance in nonaqueous lithium-oxygen (Li-O2) batteries are critically dependent on the kinetics of interfacial reactions. However, the interfacial reaction dynamic behaviors, especially the quantitative analysis, are still far from deep understanding. Using the method of laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM), we monitored the Li-O2 interfacial reaction and in situ traced the Li2O2 migration processes promoted by the solution catalyst. Different dynamic behaviors exist when regulating the concentration of the redox mediator. Quantitative analysis of the discharged Li2O2 particles shows high mobility at the early discharge stage and decayed motion in the subsequent process, indicating the solution-mediated pathway participating Li2O2 formation in the low-concentration redox mediator addition, while particles/aggregates confined into the amorphous film demonstrate simultaneous solution and surface route-mediated pathway participation in the high-concentration case. These distinctive observations of Li2O2 formation and decomposition processes present the advantage of LCM-DIM to fundamentally understand the dynamic evolution in Li-O2 batteries.

Silica-Supported Assemblage of CuII Ions with Carbon Dots for Self-Boosting and Glutathione-Induced ROS Generation

The present work introduces coordinative binding of CuII ions with both amino-functionalized silica nanoparticles (SNs) and green-emitting carbon dots (CDs) as the pregrequisite for the CuII-assisted self-assembly of the CDs at the surface of the SNs. The produced composite SNs exhibit stable in time stimuli-responsive green fluorescence derived from the CuII-assisted assemblage of CDs. The fluorescence response of the composite SNs is sensitive to the complex formation with glutathione (GSH), enabling them to detect it with the lower limit of detection of 0.15 μM. The spin-trap-facilitated electron spin resonance technique indicated that the composite SNs are capable of self-boosting generation of ROS due to CuII→CuI reduction by carbon in low oxidation states as a part of the CDs. The intensity of the ESR signals is enhanced under the heating to 38 °C. The intensity is suppressed at the GSH concentration of 0.35 mM but is enhanced at 1.0 mM of glutathione, while it is suppressed once more at the highest intracellular concentration level of GSH (10 mM). These tendencies reveal the concentrations optimal for the scavenger or reductive potential of GSH. Flow cytometry and fluorescence and confocal microscopy methods revealed efficient cell internalization of SNs-NH2-CuII-CDs comparable with that of “free” CDs.

Biochemical properties of H+-Ca2+-exchanger in the myometrium mitochondria.

Some biochemical properties of the H+-Ca2+-exchanger in uterine smooth muscle mitochondria have been described. The experiments were performed on a suspension of isolated mitochondria from the myometrium of rats. Methods of confocal microscopy, spectrofluorimetry and photon correlation spectroscopy were used. Fluo-4 probe was used to record changes in ionized Ca2+ in the matrix and cytosol; pH changes in the matrix were evaluated with BCECF. It was experimentally proved that in the myometrium instead of Na+-Ca2+-exchanger the H+-Ca2+-exchanger functions. It was activated at a physiological pH value, was carried out in stoichiometry 1: 1 and was electrogenic. The transport system was modulated by magnesium ions and the diuretic amiloride, but was not sensitive to changes in the concentration of extra-mitochondrial potassium ions. H+-Ca2+-exchanger was suppressed by antibodies against the LETM1 protein. Calmodulin may act as a regulator of H+-Ca2+-exchanger by inhibiting it.

Semi-Automated Computational Assessment of Cancer Organoid Viability Using Rapid Live-Cell Microscopy.

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess in vivo tumor tissue treatment response are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed “Apex Imaging.” We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

Visualization and quantitative analyses for mouse embryonic stem cell tracking by manipulating hierarchical data structures using time-lapse confocal microscopy images.

We present a cell tracking method for time-lapse confocal microscopy (3D) images that uses dynamic hierarchical data structures to assist cell and colony segmentation and tracking. During the segmentation, the cell and colony numbers and their geometric data are recorded for each 3D image set. In tracking, the colony correspondences between neighboring frames of time-lapse 3D images are first computed using the recorded colony centers. Then, cell correspondences in the correspondent colonies are computed using the recorded cell centers. The examples show the proposed cell tracking method can achieve high tracking accuracy for time-lapse 3D images of undifferentiated but self-renewing mouse embryonic stem (mES) cells where the number and mobility of ES cells in a cell colony may change suddenly by a colony merging or splitting, and cell proliferation or death. The geometric data in the hierarchical data structures also help the visualization and quantitation of the cell shapes and mobility.

DEVELOPMENT AND CHARACTERISATION OF 3D SOLID TUMOUR CELL MODELS FOR INDIVIDUALIZED CANCER TREATMENT

Background. To solve the problems of personalized medicine in oncology, preclinical studies based on the use of three-dimensional cellular models of tumors in vitro, including spheroids / tumoroids, are of great importance. They are an interesting tool for genetic, epigenetic, biomedical and pharmacological studies aiming to determine the most effective individual therapeutic approaches, since they allow modeling the dynamic evolution of a tumor disease from early stages to metastatic spread through interaction with the microenvironment.The purpose of the study was to compare characteristic features of formation and spatial organization of spheroids, obtained from solid malignant tumors cells with various histogenesis: melanomas, soft tissue sarcomas and osteosarcomas, epithelial tumors.Material and Methods. Solid tumor cell lines of patients who were treated from 2015 to 2021 were the basis for the creation of 3D-cell models. Fragments of tumor tissue were obtained intraoperatively: 15 samples of melanoma, 20 samples of soft tissue sarcomas and osteosarcomas, and 9 samples of epithelial tumors. All tumor cells were cultured for at least 10 passages. Methods of phase contrast, confocal microscopy, and histological techniques were used to study spheroids. Using ELISA methods and multiplex analysis, the supernatants of monolayer cell cultures and spheroids were studied for the presence of a wide range of biologically active substances that provide the processes of immunosuppression, invasion and metastasis.Results. The use of low adhesion surfaces was proven to be preferable to obtain spheroids of a given seed concentration and size of interest. The average cultivation time of spheroids was 4.7 days, and the optimal seeding concentration was 10,000 cells per well, while the spheroid diameter varied from 300 to 1000 μm depending on the type of malignant cells: the largest spheroids formed melanoma cultures. In general, the efficiency of spheroid formation was 88.6 % (39 out of 44). The introduction of fibroblasts into the 3D construct led to increasing in the invasive potential of tumor cells, which was associated with the production of IL8 (rho=0.636, p=0.035), HGF (rho=0.850, p=0.004), SCF (rho=0.857, p=0.014), FST (rho=0.685, p=0.029), Prolactin (rho=0.810, p=0.015), PECAM1 (rho=0.788, p=0.004).Conclusion. The technology of low-adhesive surfaces makes it possible to successfully create three-dimensional models of a tumor node from malignant tumors cells of various histogenesis. The colonization of a three-dimensional structure with fibroblasts enhances the biologically aggressive properties of tumor cells and demonstrates complex reciprocal interactions between the cellular elements of the tumor stroma and malignant cells, which brings the model closer to a real clinical situation.

Recent Perspectives in the Management of Fungal Keratitis.

Mycotic keratitis is common in warm, humid regions with a varying profile of pathogenic fungi according to geographical origin, socioeconomic status, and climatic condition. Clinical diagnosis can be challenging in difficult cases and those refractory to treatment. Fungal hyphae on microscopic examination and culture isolation have been the gold standard in the laboratory diagnosis of fungal keratitis. A culture isolate of the aetiological fungus is essential to perform antifungal susceptibility testing. As the culture isolation of fungi is time-consuming, causing delays in the initiation of treatment, newer investigative modalities such as in vivo confocal microscopy and molecular diagnostic methods have recently gained popularity. Molecular diagnostic techniques now help to obtain a rapid diagnosis of fungal keratitis. Genomic approaches are based on detecting amplicons of ribosomal RNA genes, with internal transcribed spacers being increasingly adopted. Metagenomic deep sequencing allows for rapid and accurate diagnosis without the need to wait for the fungus to grow. This is also helpful in identifying new emerging strains of fungi causing mycotic keratitis. A custom-tear proteomic approach will probably play an important diagnostic role in future in the management of mycotic keratitis. Positive repeat cultures are being suggested as an important gauge indicative of a poor prognosis. Positive repeat fungal cultures help to modify a treatment regimen by increasing its frequency, providing the addition of another topical and oral antifungal agent along with close follow-up for perforation and identifying need for early therapeutic keratoplasty. The role of collagen crosslinking in the treatment of fungal keratitis is not convincingly established. Rapid detection by multiplex PCR and antifungal susceptibility testing of the pathogenic fungi, adopted into a routine management protocol of fungal keratitis, will help to improve treatment outcome. Early therapy is essential in minimizing damage to the corneal tissue, thereby providing a better outcome. The role of conventional therapy with polyenes, systemic and targeted therapy of antifungal agents, newer azoles and echinocandins in fungal keratitis has been widely studied in recent times. Combination therapy can be more efficacious in comparison to monotherapy. Given the diversity of fungal aetiology, the emergence of new corneal pathogenic fungi with varying drug susceptibilities, increasing the drug resistance to antifungal agents in some genera and species, it is perhaps time to adopt recent molecular methods for precise identification and incorporate antifungal susceptibility testing as a routine.

Brillouin confocal microscopy to determine biomechanical properties of SULEEI-treated bovine pericardium for application in cardiac surgery.

Heart valves are exposed to a highly dynamic environment and underlie high tensile and shear forces during opening and closing. Therefore, analysis of mechanical performance of novel heart valve bioprostheses materials, like SULEEI-treated bovine pericardium, is essential and usually carried out by uniaxial tensile tests. Nevertheless, major drawbacks are the unidirectional strain, which does not reflect the in vivo condition and the deformation of the sample material. An alternative approach for measurement of biomechanical properties is offered by Brillouin confocal microscopy (BCM), a novel, non-invasive and three-dimensional method based on the interaction of light with acoustic waves. BCM is a powerful tool to determine viscoelastic tissue properties and is, for the first time, applied to characterize novel biological graft materials, such as SULEEI-treated bovine pericardium. Therefore, the method has to be validated as a non-invasive alternative to conventional uniaxial tensile tests. Vibratome sections of SULEEI-treated bovine pericardium (decellularized, riboflavin/UV-cross-linked and low-energy electron irradiated) as well as native and GA-fixed controls (n = 3) were analyzed by BCM. In addition, uniaxial tensile tests were performed on equivalent tissue samples and Young's modulus as well as length of toe region were analyzed from stress-strain diagrams. The structure of the extracellular matrix (ECM), especially collagen and elastin, was investigated by multiphoton microscopy (MPM). SULEEI-treated pericardium exhibited a significantly higher Brillouin shift and hence higher tissue stiffness in comparison to native and GA-fixed controls (native: 5.6±0.2 GHz; GA: 5.5±0.1 GHz; SULEEI: 6.3±0.1 GHz; n = 3, p < 0.0001). Similarly, a significantly higher Young's modulus was detected in SULEEI-treated pericardia in comparison to native tissue (native: 30.0±10.4 MPa; GA: 31.8±10.7 MPa; SULEEI: 42.1±7.0 MPa; n = 3, p = 0.027). Native pericardia showed wavy and non-directional collagen fibers as well as thin, linear elastin fibers generating a loose matrix. The fibers of GA-fixed and SULEEI-treated pericardium were aligned in one direction, whereat the SULEEI-sample exhibited a much denser matrix. BCM is an innovative and non-invasive method to analyze elastic properties of novel pericardial graft materials with special mechanical requirements, like heart valve bioprostheses.

Tailoring of silica nanoarchitecture to optimize Cu(2−x)S based image-guided chemodynamic therapy agent

The present work introduces various synthetic and post-synthetic modes of co-doping of oleate-oleylamine-stabilized Cu(2−x)S cores with red emitting [Ru(dipy)3]2+ into amino-modified silica nanoparticles (SNs) with the aim to combine chemodynamic therapy (CDT) with cellular uptake and imaging functions. Thereto, the ROS generation by the Cu(2−x)S embedded into the SNs was manifested by the spin trap facilitated ESR technique and correlated with such parameters as size of the embedded cores, content and oxidation extent of copper ions. The parameters were varied through the different extent of an oxidative degradation of the embedded Cu(2−x)S cores by their post-treating with histidine, polyethylenimine and citrate-stabilized carbon dots (CDs). The treating by CDs was chosen as the optimal post-synthetic modification of the composite SNs due to combination of CDT with high stability to aggregation. The green or dual green-red emission of the CD-treated composite SNs visualizes their cell internalization and intracellular localization by means of fluorescence and confocal microscopy methods. Correlation of cytotoxicity data of the differently treated composite SNs with their ability to ROS generation and the intracellular localization highlight the enhanced cell internalization and nuclear confinement of the CD-treated composite SNs as the factor enhancing their cytotoxicity in greater extent than the CDT function.

Impact of Algicidal Bacillus mycoides on Diatom Ulnaria acus from Lake Baikal

Algae–bacteria interactions play an important role in water ecosystems. In this work, the BS2-15 algicidal strain was isolated from the bottom sediments of Lake Baikal and identified as Bacillus mycoides on the basis of 16S rDNA sequencing, its described ultrastructure, and biochemical properties. B. mycoides BS2-15 was demonstrated to have a strong algicidal effect against a freshwater diatom culture of Ulnaria acus, inhibiting its growth and increasing frustules fragility. By analyzing the impact of bacterial filtrate onto the cells of U. acus, we demonstrated that perhaps an algicidal compound is produced by bacteria independently in the presence of diatoms in a medium. Using methods of TUNEL and confocal microscopy, we revealed that the bacterial algicidal effect on the diatom cells results in DNA fragmentation, nucleus destruction, and neutral lipid accumulation. This phenomenon highlights the complexity of algae–bacteria interactions and their potential role in regulating water ecosystem microbial populations.

Correlative Light-Environmental Scanning Electron Microscopy of Plasma Membrane Efflux Carriers of Plant Hormone Auxin.

Fluorescence light microscopy provided convincing evidence for the domain organization of plant plasma membrane (PM) proteins. Both peripheral and integral PM proteins show an inhomogeneous distribution within the PM. However, the size of PM nanodomains and protein clusters is too small to accurately determine their dimensions and nano-organization using routine confocal fluorescence microscopy and super-resolution methods. To overcome this limitation, we have developed a novel correlative light electron microscopy method (CLEM) using total internal reflection fluorescence microscopy (TIRFM) and advanced environmental scanning electron microscopy (A-ESEM). Using this technique, we determined the number of auxin efflux carriers from the PINFORMED (PIN) family (NtPIN3b-GFP) within PM nanodomains of tobacco cell PM ghosts. Protoplasts were attached to coverslips and immunostained with anti-GFP primary antibody and secondary antibody conjugated to fluorochrome and gold nanoparticles. After imaging the nanodomains within the PM with TIRFM, the samples were imaged with A-ESEM without further processing, and quantification of the average number of molecules within the nanodomain was performed. Without requiring any post-fixation and coating procedures, this method allows to study details of the organization of auxin carriers and other plant PM proteins.

Distinct Cellular Profiles of Hif1a and Vegf mRNA Localization in Microglia, Astrocytes and Neurons during a Period of Vascular Maturation in the Auditory Brainstem of Neonate Rats

Defining the relationship between vascular development and the expression of hypoxia-inducible factors (Hifs) and vascular endothelial growth factor (Vegf) in the auditory brainstem is important to understand how tissue hypoxia caused by oxygen shortage contributes to sensory deficits in neonates. In this study, we used histology, molecular labeling, confocal microscopy and 3D image processing methods to test the hypothesis that significant maturation of the vascular bed in the medial nucleus of the trapezoid body (MNTB) occurs during the postnatal period that precedes hearing onset. Isolectin-B4 histochemistry experiments suggested that the MNTB vasculature becomes more elaborate between P5 and P10. When combined with a cell proliferation marker and immunohistochemistry, we found that vascular growth coincides with a switch in the localization of proliferating cells to perivascular locations, and an increase in the density of microglia within the MNTB. Furthermore, microglia were identified as perivascular cells with proliferative activity during the period of vascular maturation. Lastly, combined in situ hybridization and immunohistochemistry experiments showed distinct profiles of Hif1a and Vegf mRNA localization in microglia, astrocytes and MNTB principal neurons. These results suggest that different cells of the neuro-glio-vascular unit are likely targets of hypoxic insult in the auditory brainstem of neonate rats.

Application of Nanotechnologies in Studying Yeast Structure in Candida

This review considers the methods of probe, electron, and confocal microscopy for studying the structure of the yeast Candida. The latest advances in the study of antimicrobial effects by high-resolution microscopy are presented. The advantages and disadvantages of each method are presented, as well as the limits of their application. Processes such as cell wall lysis and destruction of the cytoplasmic membrane caused by antifungal drugs are described, as well as their effect on the nanostructural or mechanical properties of Candida. Particular attention is paid to sample preparation of Candida and scanning protocols.

Starch Granules in Arabidopsis thaliana Mesophyll and Guard Cells Show Similar Morphology but Differences in Size and Number.

Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.

Artocarpus integrifolia seed lectin enhances membrane damage, oxidative stress and biofilm inhibition activity of silver nanoparticles against Staphylococcus aureus

Functionalizing the nanoparticles to target the bacteria surface is an effective approach to curb drug resistance pathogens. The present study describes the synthesis of N-myristoyltaurine capped silver nanoparticles (AgNPs) and functionalization of AgNPs with jacalin (JAgNPs), a lectin from the seeds of Artocarpus integrifolia . JAgNPs has fourfold lower minimum inhibitory concentration than AgNPs and kills the S. aureus in less than 30 min through inducing oxidative stress and membrane damage. JAgNPs (31.25 µM) was found to have excellent biofilm inhibitory activity at lower concentration than AgNPs (62.5 µM). This was judged by crystal violet assay, light microscopy imaging and confocal laser scanning microscopy (CLSM) method. CLSM analysis reveals that JAgNPs is highly effective as compared to AgNPs in reducing the bio-volume and average biofilm thickness to 18.3 ± 2.5 µm 3 /µm 2 and 14.0 ± 2.6 µm, respectively. Scanning electron microscopy (SEM) study reveals that JAgNPs prevents the colony formation and inhibited exopolysaccharide synthesis, thereby reducing the formation of biofilm. At minimum biofilm inhibitory concentrations, SEM and double staining technique using CLSM provides evidence that cells are intact and live, suggesting that NPs exhibit true biofilm activity. The findings indicate that jacalin functionalization may offer a unique way to use nanosilver at lower concentrations for treating infection associated with S. aureus biofilm. • Jacalin functionalized AgNPs kills S. aureus in less than 30 min. • NPs exert antibacterial activity through inducing oxidative stress and membrane damage. • Jacalin-AgNPs exhibit antibiofilm activity at 0.5X MIC. • NPs inhibits exopolysaccharide biosynthesis.

Confocal microscopy 3D imaging of diesel particulate matter

To date, diesel particulate matter (DPM) has been described as aggregates of spherule particles with a smooth appearing surface. We have used a new colour confocal microscope imaging method to study the 3D shape of diesel particulate matter (DPM); we observed that the particles can have sharp jagged appearing edges and consistent with these findings, 2D light microscopy demonstrated that DPM adheres to human lung epithelial cells. Importantly, the slide preparation and confocal microscopy method applied avoids possible alteration to the particles’ surfaces and enables colour 3D visualisation of the particles. From twenty-one PM10 particles, the mean (standard deviation) major axis length was 5.6 (2.25) μm with corresponding values for the minor axis length of 3.8 (1.25) μm. These new findings may help explain why air pollution particulate matter (PM) has the ability to infiltrate human airway cells, potentially leading to respiratory tract, cardiovascular and neurological disease.

Increased Incidence and Plasma-Biofilm Formation Ability of SCCmec Type IV Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated From Patients With Bacteremia.

In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected by liposuction surgery, was digested with collagenase to separate adipocytes from other tissue cells, comprising blood and lymphatic endothelial cells, fibroblasts, and all vessel- and tissue-resident leukocytes - the stromal vascular fraction (SVF). SVF cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, adding Brefeldin-A to prevent cytokine secretion during the final 4 hours. Cells were incubated with CD11b-FITC and CD40-APC (M1 MØ)‚ or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then incubated with either (1) anti-TNF-PE, (2) anti- IL-1β-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGFβ-PE or (6) isotype-IgG-PE (control), and stained with Hoechst 33342, preserved in permanent mounting media and examined by confocal microscopy. The FITC, PE and APC fluorescence channels were set to achieve minimal cross-channel emission using single-colour controls and voltages set for optimal detection by thresholding on isotype-IgG stained activated cells. Finally, transmission and z-stack images were captured. Cells were analysed as regions of interest (ROI) based on Hoechst-33342 then enumerated as FITC+, FITC+APC+ or FITC+APC+PE+ using an ImageJ script and exported into Excel. This permitted the examination of >9000 SVF cells individually, per LE sample. This method allows for the analysis of a high number of heterogeneous cells defined into any subtype or combination by the investigators' choice of surface and intracellular expression profiles. Fibroblasts, or other cytokine producing cells, can also be analysed by using other antibodies, and the cell count data can be correlated with any clinical or laboratory data.

Роль іонів калію у біосинтезі оксиду азоту в мітохондріях гладенького м’яза

The NO-synthase activity (mtNOS) in mitochondria of uterine smooth muscle was studied. The mitochondrial localization of NO synthesis in myocytes was proved using laser confocal microscopy method and specific fluorescent probes MitoTracker Orange (specific to mitochondria) and DAFFM (NO-sensitive fluorescent probe). It was demonstrated using flow cytometry that nitric oxide biosynthesis in isolated mytochondria decreased in the presence of a constitutive NOsynthase blocker 2-aminopyridine (100 μmol per l, 50% inhibition) and monoclonal antibodies (2.5 μg anti-Let m1 per 50 μg protein) against the H+-Ca2+-exchanger (Letm1 protein), but was’t sensitive to the mitochondrial permeability transition pore inhibitor cyclosporin A (5 μmol per l). A decrease of potassium ions concentration in the incubation medium and the presence of various types of potassium channel inhibitors significantly inhibited the NO-synthase reaction. We have concluded that potassium permeability of the inner mitochondrial membrane plays important role in the regulation of mtNOS activity.