wide-field microscopy
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Artifact-free microscopic images represent a key requirement of multi-parametric image analysis in modern biomedical research. Holography microscopy (HM) is one of the quantitative phase imaging techniques, which has been finding new applications in life science, especially in morphological screening, cell migration, and cancer research. Rather than the classical imaging of absorbing (typically stained) specimens by bright-field microscopy, the information about the light-wave's phase shifts induced by the biological sample is employed for final image reconstruction. In this comparative study, we investigated the usability and the reported advantage of the holography imaging. The claimed halo-free imaging was analyzed compared to the widely used Zernike phase-contrast microscopy. The intensity and phase cross-membrane profiles at the periphery of the cell were quantified. The intensity profile for cells in the phase-contrast images suffers from the significant increase in intensity values around the cell border. On the contrary, no distorted profile is present outside the cell membrane in holography images. The gradual increase in phase shift values is present in the internal part of the cell body projection in holography image. This increase may be related to the increase in the cell internal material according to the dry mass theory. Our experimental data proved the halo-free nature of the holography imaging, which is an important prerequisite of the correct thresholding and cell segmentation, nowadays frequently required in high-content screening and other image-based analysis. Consequently, HM is a method of choice whenever the image analysis relies on the accurate data on cell boundaries.
- MeSH
- artefakty MeSH
- HeLa buňky MeSH
- holografie * MeSH
- lidé MeSH
- mikroskopie fázově kontrastní * MeSH
- nádorové buňky kultivované MeSH
- Saccharomyces cerevisiae cytologie růst a vývoj MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.
- MeSH
- chromozomy rostlin chemie genetika metabolismus MeSH
- DNA-topoisomerasy typu II metabolismus MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční mikroskopie metody MeSH
- indoly chemie MeSH
- ječmen (rod) cytologie genetika MeSH
- konfokální mikroskopie metody MeSH
- metafáze genetika MeSH
- reprodukovatelnost výsledků MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
The magnetic domain patterns of amorphous bilayered FeSiB/FeNbSiB and FeNbCuSiB/CoSiB ribbons are observed and analysed using the magneto-optical Kerr microscopy (MOKM) and magnetic force microscopy (MFM). Both microscopic techniques are highly sensitive to the sample surface; possibility of Kerr microscopy to visualize the domains separately in both layers is achieved by focusing the laser spot on the ribbon cross section. Wide curved domains as well as fine fingerprint domains were detected at the surface of ribbons due to presence of local stresses coming from the preparation process. With respect to high lateral resolution of MFM and its out-of-plane magnetization sensitivity, the perpendicularly magnetized crossed stripe domain patterns can be selected as well. Coiling of the ribbons on the half-round-end sample holder is often used to induce and control the magnetic anisotropy of these alloys. Changes in the magnetic domain structure at the outer-coiled surface and its dependence on the sign of magnetostriction coefficient are discussed in detail. Finally, the MFM images in the presence of external in-plane magnetic field up to ±40 kA/m are shown.
- Publikační typ
- časopisecké články MeSH
Enzymes have a wide range of applications in different industries owing to their high specificity and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and under process conditions are still largely uncertain. Here, three microscopy techniques - bright-field, confocal and electron microscopy - were applied to determine the distribution and structure of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological changes that strongly correlated with increased catalytic activity compared to less structured, non-treated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions.
- MeSH
- biokatalýza MeSH
- dioxany farmakologie MeSH
- furany farmakologie MeSH
- hydrolasy chemie metabolismus MeSH
- mikroskopie metody MeSH
- proteiny červů chemie metabolismus MeSH
- Schistosoma japonicum enzymologie MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Oxidative stress is involved in a number of pathological conditions and the generated oxidatively modified lipids influence membrane properties and functions, including lipid-protein interactions and cellular signaling. Brewster angle microscopy demonstrated oxidatively truncated phosphatidylcholines to promote phase separation in monolayers of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), sphingomyelin (SM) and cholesterol (Chol). More specifically, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), was found to increase the miscibility transition pressure of the SM/Chol-phase. Lateral diffusion of lipids is influenced by a variety of membrane properties, thus making it a sensitive parameter to observe the coexistence of different lipid phases, for instance. The dependence on lipid lateral packing of the lateral diffusion of fluorophore-containing phospholipid analogs was investigated in Langmuir monolayers composed of POPC, SM, and Chol and additionally containing oxidatively truncated phosphatidylcholines, using fluorescence correlation spectroscopy (FCS). To our knowledge, these are the first FCS results on miscibility transition in ternary lipid monolayers, confirming previous results obtained using Brewster angle microscopy on such lipid monolayers. Wide-field fluorescence microscopy was additionally employed to verify the transition, i.e. the loss and reformation of SM/Chol domains.
- MeSH
- algoritmy MeSH
- chemické modely MeSH
- cholesterol chemie MeSH
- difuze MeSH
- fosfatidylcholiny chemie MeSH
- fosfolipidy chemie MeSH
- mikroskopie metody MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- sfingomyeliny chemie MeSH
- unilamelární lipozómy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The evaluation of large experimental datasets is a fundamental aspect of research in every scientific field. Streamlining this process can improve the reliability of results while making data analysis more efficient and faster to execute. In biomedical research it is often very important to determine the type of cell death after various treatments. Thus, differentiating between viable, apoptotic, and necrotic cells provide critical insights into the treatment efficacy, a key aspect in the field of drug development. Fluorescent microscopy is perceived as a widely used technique for cell metabolism assessment and can therefore be used to investigate treatment outcomes after staining samples with cell death detection kit. However, accurate evaluation of therapeutic results requires quantitative analysis, often necessitating extensive postprocessing of imaging data. In this study, we introduce a complementary tool designed as a macro for the Fiji platform, enabling the automated postprocessing of fluorescent microscopy images to accurately distinguish and quantify viable, apoptotic, and necrotic cells.
- MeSH
- apoptóza MeSH
- buněčná smrt MeSH
- fluorescenční mikroskopie * metody MeSH
- lidé MeSH
- nekróza MeSH
- počítačové zpracování obrazu * metody MeSH
- software MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Margaritaella gracilis gen. n. et sp. n. (Proteocephalidea: Proteocephalinae) found in the intestine of Callichthys callichthys (Linnaeus) from the Paraná River basin is described. The new genus is placed in the Proteocephalinae because of the medullary position of the genital organs. It differs from all known genera included in the Proteocephalinae by the following combination of characters: 1) scolex with a conspicuous cluster of drop-shaped gland cells posterior to suckers; 2) strobila with a low number of proglottides, all much longer than wide; 3) testes arranged in one field, composed of two parallel rows of testes separated by the uterus; 4) ovary delicate, H-shaped, with branches slender and deeply folliculate at the edges, located at 25–35% from the posterior end; 5) uterus largely extended posterior to the ovary but not reaching the end of proglottis; and 6) vitelline follicles in two narrow lateral bands, largely extended posterior to the ovary. Scanning electron microscopy revealed three types of microtriches on the tegument surface: acicular and capiliform filitriches and gladiate spinitriches. The relative size of the ovary, a character recently used in the systematics of the proteocephalidean cestodes, was calculated for the new species and compared with other species of the group. M. gracilis is the first record of a proteocephalidean cestode parasitizing a callichthyid catfish.
- MeSH
- Cestoda anatomie a histologie klasifikace ultrastruktura MeSH
- cestodózy epidemiologie parazitologie veterinární MeSH
- mikroskopie elektronová rastrovací veterinární MeSH
- nemoci ryb epidemiologie parazitologie MeSH
- řeky MeSH
- sumci parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Argentina MeSH
Cells continuously communicate with the surrounding environment employing variety of signaling molecules and pathways to integrate and transport the information in the cell. An example of signaling initiation is binding of extracellular ligand to its receptor at the plasma membrane. This initializes enzymatic reactions leading to the formation of bi- or multimolecular signaling complexes responsible for the regulation or progress of signal transduction. Here, we describe three imaging techniques enabling detection of individual signaling molecules, their complexes, and clusters in human cells. Described imaging techniques require only basic microscopy systems available in the majority of current biomedical research centers but apply advanced data processing. First, total internal reflection fluorescence microscopy (TIRFM) variant of wide-field fluorescence microscopy for imaging highly dynamic clusters is described. Second, superresolution localization microscopy techniques-photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-recently enabled to achieve higher resolution with precision limit of about 20 nm in fixed samples. The developments toward live cell superresolution imaging are indicated. Third, raster image correlation spectroscopy (RICS) employed for molecular diffusion and binding analysis explains the advantages and hurdles of this novel method. Presented techniques provide a new level of detail one can learn about higher organization of signaling events in human cells.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčný tracking metody MeSH
- fluorescenční barviva MeSH
- fluorescenční mikroskopie metody MeSH
- interpretace obrazu počítačem metody MeSH
- Jurkat buňky MeSH
- lidé MeSH
- ligandy MeSH
- mezibuněčná komunikace MeSH
- signální transdukce MeSH
- zelené fluorescenční proteiny MeSH
- zobrazování trojrozměrné MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Coronary artery development is an intensely studied field. Mice are a popular genetic model for developmental studies, but there is no widely accepted protocol for high-throughput, high-resolution imaging of their developmental and adult coronary artery anatomy. RESULTS: Using tissue-clearing protocols and confocal microscopy, we have analyzed embryonic and juvenile mouse hearts in Cx40:GFP knock-in models with a special focus on septal artery development. We found that the septal artery, which supplies the interventricular septum, was initially formed as an arterial plexus that connected to both the left and right coronary arteries. During development, the plexus was remodeled into a single tube, which then remained connected only to the right coronary artery. Since optical imaging became limited at postnatal stages, it was supplemented with injection techniques using India ink and Microfil; the latter was subsequently analyzed with micro-CT to visualize the anatomy of coronary vessels in 3D. CONCLUSIONS: The techniques described here enable us to study the finer details of coronary artery development in mice and can, therefore, be implemented to study the pathogenesis of coronary malformations in various mouse models. Developmental Dynamics 247:1018-1027, 2018. © 2018 Wiley Periodicals, Inc.
