3D confocal analysis
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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.
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
A well developed capillary bed is essential for proper function of skeletal muscles. We present for the first time a triple immunofluorescent method suitable for staining capillaries and muscle fibre outlines in thick sections of human skeletal muscle, applying antibodies against collagen IV (in red) and F8 (in green) as well as Ulex europaeus lectin, visualized in green fluorescence. Further, we present possibilities for quantitative evaluation of the capillary network which implies the length of capillaries per unit volume of muscle tissue (Lcap/Vmuscle) and the length of capillaries supplying individual muscle fibres per unit fibre length (Lcap/Lfib), per surface area (Lcap/Sfib) and per volume (Lcap/Vfib) as well as the course of capillaries in the muscle. The latter can be described by the tortuosity, orientation and mean capillary length. To get reasonable results we met the following requirements: i) high quality thick tissue sections, from which 3D image data were acquired; ii) immunofluorescent methods suitable for confocal microscopy; iii) penetration of the fluorescent dyes throughout the tissue section; iv) proper 3D image analysis methods for performing reliable measurements and v) control over relevant tissue deformations. The developed methodology is illustrated by results obtained from autopsy or biopsy samples of three human muscles, i.e. vastus lateralis, multifidus and masseter muscle that exhibit differences in genetic background, innervation, tasks and functional activity.
- MeSH
- barvení a značení metody MeSH
- čtyřhlavý sval stehenní anatomie a histologie krevní zásobení MeSH
- dospělí MeSH
- fluorescenční mikroskopie MeSH
- konfokální mikroskopie MeSH
- kosterní svaly anatomie a histologie krevní zásobení MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikrocévy anatomie a histologie MeSH
- monoklonální protilátky imunologie metabolismus MeSH
- musculus masseter anatomie a histologie krevní zásobení MeSH
- počítačové zpracování obrazu metody MeSH
- rostlinné lektiny metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- zobrazování trojrozměrné metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We present a complex approach to volume reconstruction of large tissue specimens. It consists of embedding the specimen into a paraffi n block and using this non-deparaffi nized specimen for reconstruction; Cutting the specimen into physical slices with simultaneous recording pictures from the cutting plane of the block by a USB light microscope to obtain a-priori image information about the original size and shape of the specimen; Acquisition of data by a confocal microscope; Composition of a high-resolution image from confocal data by using rigid-body and elastic registration techniques incorporating images captured by the USB light microscope to preserve original shape and size of the specimen.
- MeSH
- experimenty na zvířatech MeSH
- financování organizované MeSH
- klinické laboratorní techniky přístrojové vybavení využití MeSH
- konfokální mikroskopie metody přístrojové vybavení využití MeSH
- počítačové zpracování obrazu metody využití MeSH
- validace softwaru MeSH
- zalévání tkání do parafínu metody využití MeSH
- zobrazování trojrozměrné metody přístrojové vybavení využití MeSH
- zvířata MeSH
- Check Tag
- zvířata 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
Neuronal activity and many pathological states in the CNS are accompanied by transient astrocytic swelling, which affects excitability, extrasynaptic transmission, and neuron-glia interactions. By using three-dimensional confocal morphometry (3DCM), we quantified the morphometric parameters of astrocytes in intact tissue. In experiments performed in brain cortex slices from transgenic GFAP/EGFP mice, we applied 3DCM to study the dynamic changes in astrocyte morphology during hypotonic stress. Our morphometric analysis showed that the effect of a 10-min application of hypotonic solution (200 mmol/kg) on the swelling of different cell compartments was dependent on the extent of the swelling of the total astrocyte volume. If the swelling of the whole cell, i.e., soma and processes, was less than approximately 10%, there were no differences between the swelling of the soma and the processes. However, if the swelling of the total cell volume was greater than 10%, the swelling of the processes was greater than the swelling of the soma. Analyzing the effect of hypotonic solution on the morphology of these astrocytes revealed that the total cell volume increased; however, certain cell compartments were distinguished in which the volume increased, whereas in other compartments cell volume decreased or apparently did not change, and the structure of some compartments was altered. Our data show that astrocytes in brain slices undergoing hypotonic stress display cell volume regulation as well as transient changes in morphology.
- MeSH
- astrocyty ultrastruktura MeSH
- financování organizované MeSH
- gliový fibrilární kyselý protein genetika MeSH
- hypotonické roztoky MeSH
- konfokální mikroskopie MeSH
- mozek ultrastruktura MeSH
- myši transgenní MeSH
- myši MeSH
- orgánové kultury - kultivační techniky MeSH
- počítačové zpracování obrazu MeSH
- promotorové oblasti (genetika) MeSH
- zelené fluorescenční proteiny genetika MeSH
- zobrazování trojrozměrné MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
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
A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy-to-apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17-day-old laboratory Wistar rat embryo. (c) 2008 Wiley-Liss, Inc.
- MeSH
- algoritmy MeSH
- embryo savčí cytologie MeSH
- financování organizované MeSH
- konfokální mikroskopie metody MeSH
- krysa rodu rattus MeSH
- mikrotomie metody MeSH
- počítačové zpracování obrazu metody MeSH
- pružnost MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
