3D microscopy
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Super -rozlišovací mikroskopie je schopna různými způsoby obejít difrakční limit a dosahovat izotropického rozlišení desítek nanometrů. V současné době se vyvíjí značné úsilí, aby stejná kvalita výsledků, které lze získat s fixovanými vzorky, byla dosažitelná i s buňkami živými. Vzhledem k četnosti dynamických vnitrobuněčných procesů je žádoucí, aby super -rozlišovací 3D mikroskopie byla co nejdříve běžným a dostupným způsobem, jak tyto procesy pozorovat a zkoumat. Mitochondrie, semiautonomní buněčné elektrárny, skrývají stále spoustu nezodpovězených otázek, na které by v budoucnu právě 3D super -rozlišovací mikroskopie živých buněk mohla nalézt odpovědi.
Super -resolution microscopy can variously circumvent the Abbe's diffraction limit and achieve an isotropic resolution of tens of nanometers. Considerable efforts are currently being made to ensure that the same quality of results that can be obtained using fixed samples are achievable using living cells. Since the numerous intracellular dynamic processes occur in cells, it is desirable to make super -resolution 3D microscopy an available method as soon as possible in order to investigate these processes. Mitochondria, semi -autonomous cellular power -plants, still hide a lot of unanswered questions, to which specifically 3D super -resolution microscopy of living cells could find answers in the future.
Cíl: Cílem sdělení je seznámit čtenáře s aktuální situací v možnostech využití 3D skenerů ve stomatologických a ortodontických praxích. Úvod: Digitalizace modelů přináší nespočet výhod, ať už se jedná o měření na modelech, komunikaci postupu léčby s jinými odborníky, skladování modelů či jejich zhotovování. Materiál a metodika: Laboratorní a intraorální skenery dostupné na současném trhu. Výsledky: Přesnost a vysoká úroveň technických parametrů všech popsaných skenerů je příznivá pro použití v lékařské praxi. Z laboratorních skenerů je u ortodontistů v České republice oblíbená značka 3Shape pro její rychlé snímání. Z intraorálních skenerů je nejvíce využívaný skener Trios (3Shape) a nyní čím dál více iTero® pro jeho přímou návaznost na systém Invisalign. Závěr: 3D skenery jsou pro ortodontisty praktickými pomocníky a pro pacienty příjemným komfortem. Rozšiřují a usnadňují odbornou komunikaci a jsou také prostředkem pro názornou demonstraci postupu a variant terapie pro konkrétního pacienta. V průběhu několika let se nepochybně stanou běžnou součástí většiny stomatologických praxí.
Objective: The aim of the paper is to familiarize the reader with the current status in the possibilities of using 3D scanners in dental and orthodontic practices. Introduction: The digitization of models brings countless benefits, whether it is measurement on models, communication of treatment with other professionals, storage or making them. Material and methodology: Laboratory and intraoral scanners available in the current market. Results: The accuracy and high level of technical parameters of all the scanners described is favorable for use in medical practice. From the laboratory scanners, the 3Shape is a favorite for orthodontists in the Czech Republic for its fast scanning. From intraoral scanners, Trios (3Shape) is the most widely used scanner, and now more and more iTero® for its direct connection to the Invisalign system. Conclusion: 3D scanners are practical helpers for the orthodontists, for patients‘ pleasurable comfort. They are expanding and facilitating professional communication, and they are also a means of illustrating the progress and variants of therapy for a particular patient. Over the course of several years, they will undoubtedly become a common part of most dental practices.
- MeSH
- design s pomocí počítače přístrojové vybavení MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- mandibula diagnostické zobrazování MeSH
- maxila diagnostické zobrazování MeSH
- odontometrie přístrojové vybavení MeSH
- optická koherentní tomografie MeSH
- ortodoncie * přístrojové vybavení MeSH
- ortodontické přístroje - design metody přístrojové vybavení MeSH
- plánování péče o pacienty MeSH
- počítačová simulace * MeSH
- počítačové zpracování obrazu přístrojové vybavení MeSH
- zobrazování trojrozměrné * přístrojové vybavení MeSH
- zubní modely * MeSH
- zubní technika otisková přístrojové vybavení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Reliable 3D detection of diffraction-limited spots in fluorescence microscopy images is an important task in subcellular observation. Generally, fluorescence microscopy images are heavily degraded by noise and non-specifically stained background, making reliable detection a challenging task. In this work, we have studied the performance and parameter sensitivity of eight recent methods for 3D spot detection. The study is based on both 3D synthetic image data and 3D real confocal microscopy images. The synthetic images were generated using a simulator modeling the complete imaging setup, including the optical path as well as the image acquisition process. We studied the detection performance and parameter sensitivity under different noise levels and under the influence of uneven background signal. To evaluate the parameter sensitivity, we propose a novel measure based on the gradient magnitude of the F1 score. We measured the success rate of the individual methods for different types of the image data and found that the type of image degradation is an important factor. Using the F1 score and the newly proposed sensitivity measure, we found that the parameter sensitivity is not necessarily proportional to the success rate of a method. This also provided an explanation why the best performing method for synthetic data was outperformed by other methods when applied to the real microscopy images. On the basis of the results obtained, we conclude with the recommendation of the HDome method for data with relatively low variations in quality, or the Sorokin method for image sets in which the quality varies more. We also provide alternative recommendations for high-quality images, and for situations in which detailed parameter tuning might be deemed expensive.
3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic β-cells, INS-1E, MFD between F1α were ~80 nm at 0 and 3 mM glucose, whereas decreased to 61 nm and 57 nm upon glucose-stimulated insulin secretion (GSIS) at 11 mM and 20 mM glucose, respectively. Shorter F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α correlated to average 20 nm and 15 nm cristae width at 0 and 3 mM glucose, and 9 nm or 8 nm after higher glucose simulating GSIS (11, 20 mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100 nm) and average distances between nucleoids (~1000 nm). Double channel PALM/dSTORM with Eos-lactamase-β plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.
- MeSH
- beta-buňky cytologie metabolismus MeSH
- buňky Hep G2 MeSH
- DNA vazebné proteiny metabolismus MeSH
- fluorescenční mikroskopie přístrojové vybavení MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mitochondriální DNA chemie metabolismus MeSH
- mitochondriální membrány metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- potkani Wistar MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.
Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.
3D microscopy and image analysis provide reliable measurements of length, branching, density, tortuosity and orientation of tubular structures in biological samples. We present a survey of methods for analysis of large samples by measurement of local differences in geometrical characteristics. The methods are demonstrated on the structure of the capillary bed in a rat brain.
- MeSH
- arteriae cerebrales cytologie MeSH
- fluorescenční mikroskopie metody MeSH
- interpretace obrazu počítačem metody MeSH
- kapiláry cytologie MeSH
- konfokální mikroskopie metody MeSH
- krysa rodu rattus MeSH
- vylepšení obrazu metody MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.
- MeSH
- blízká infračervená spektroskopie metody MeSH
- fibrosarkom patologie MeSH
- invazivní růst nádoru MeSH
- lidé MeSH
- mikroskopie elektronová rastrovací metody MeSH
- nádorové buňky kultivované MeSH
- nádory prsu patologie MeSH
- počítačové zpracování obrazu MeSH
- podozomy patologie MeSH
- zobrazování trojrozměrné metody MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.
- MeSH
- barvení a značení MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- organoidy * MeSH
- prsy MeSH
- zobrazování trojrozměrné * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.
- MeSH
- kapiláry anatomie a histologie ultrastruktura MeSH
- konfokální mikroskopie metody MeSH
- kosterní svalová vlákna ultrastruktura MeSH
- kosterní svaly krevní zásobení cytologie MeSH
- krysa rodu rattus MeSH
- počítačové zpracování obrazu MeSH
- potkani Wistar MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
