Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

• MeSH
• design vybavení metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• jehly MeSH
• optická vlákna MeSH
• optické prostředky MeSH
• osvětlení metody   MeSH
• světlo MeSH
• zobrazování trojrozměrné metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer's disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.

• MeSH
• anestezie MeSH
• arteria cerebri media fyziologie   MeSH
• arterioly fyziologie   MeSH
• fluorescenční mikroskopie metody   MeSH
• glymfatický systém fyziologie   MeSH
• mozek ultrastruktura   MeSH
• mozkomíšní mok metabolismus   MeSH
• mozkový krevní oběh fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• neurozobrazování metody   MeSH
• spánek fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Long-term fluorescence live-cell imaging experiments have long been limited by the effects of excitation-induced phototoxicity. The advent of light-sheet microscopy now allows users to overcome this limitation by restricting excitation to a narrow illumination plane. In addition, light-sheet imaging allows for high-speed image acquisition with uniform illumination of samples composed of multiple cell layers. The majority of studies conducted thus far have used custom-built platforms with specialized hardware and software, along with specific sample handling approaches. The first versatile commercially available light-sheet microscope, Lightsheet Z.1, offers a number of innovative solutions, but it requires specific strategies for sample handling during long-term imaging experiments. There are currently no standard procedures describing the preparation of plant specimens for imaging with the Lightsheet Z.1. Here we describe a detailed protocol to prepare plant specimens for light-sheet microscopy, in which Arabidopsis seeds or seedlings are placed in solid medium within glass capillaries or fluorinated ethylene propylene tubes. Preparation of plant material for imaging may be completed within one working day.

• MeSH
• Arabidopsis MeSH
• fluorescenční mikroskopie * MeSH
• rostlinné buňky * MeSH
• vývoj rostlin * MeSH
• zalévání tkání metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo-devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo-devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-α-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.

• Publikační typ
• časopisecké články MeSH

Mitotic cell division in plants is a dynamic process playing a key role in plant morphogenesis, growth, and development. Since progress of mitosis is highly sensitive to external stresses, documentation of mitotic cell division in living plants requires fast and gentle live-cell imaging microscopy methods and suitable sample preparation procedures. This chapter describes, both theoretically and practically, currently used advanced microscopy methods for the live-cell visualization of the entire process of plant mitosis. These methods include microscopy modalities based on spinning disk, Airyscan confocal laser scanning, structured illumination, and light-sheet bioimaging of tissues or whole plant organs with diverse spatiotemporal resolution. Examples are provided from studies of mitotic cell division using microtubule molecular markers in the model plant Arabidopsis thaliana, and from deep imaging of mitotic microtubules in robust plant samples, such as legume crop species Medicago sativa.

• MeSH
• Arabidopsis metabolismus   fyziologie   MeSH
• mikroskopie metody   MeSH
• mikrotubuly fyziologie   MeSH
• mitóza fyziologie   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• elektronová mikroskopie MeSH
• fyzika MeSH
• mikroskopie MeSH
• Publikační typ
• monografie MeSH

• Konspekt
• Fyzika
• NLK Obory
• fyzika, biofyzika

Head and neck squamous cell carcinoma is one of the most aggressive tumours and is typically diagnosed too late. Late diagnosis requires an urgent decision on an effective therapy. An individualized test of chemosensitivity should quickly indicate the suitability of chemotherapy and radiotherapy. No ex vivo chemosensitivity assessment developed thus far has become a part of general clinical practice. Therefore, we attempted to explore the new technique of coherence-controlled holographic microscopy to investigate the motility and growth of live cells from a head and neck squamous cell carcinoma biopsy. We expected to reveal behavioural patterns characteristic for malignant cells that can be used to imrove future predictive evaluation of chemotherapy. We managed to cultivate primary SACR2 carcinoma cells from head and neck squamous cell carcinoma biopsy verified through histopathology. The cells grew as a cohesive sheet of suspected carcinoma origin, and western blots showed positivity for the tumour marker p63 confirming cancerous origin. Unlike the roundish colonies of the established FaDu carcinoma cell line, the SACR2 cells formed irregularly shaped colonies, eliciting the impression of the collective invasion of carcinoma cells. Time-lapse recordings of the cohesive sheet activity revealed the rapid migration and high plasticity of these epithelial-like cells. Individual cells frequently abandoned the swiftly migrating crowd by moving aside and crawling faster. The increasing mass of fast migrating epithelial-like cells before and after mitosis confirmed the continuation of the cell cycle. In immunofluorescence, analogously shaped cells expressed the p63 tumour marker, considered proof of their origin from a carcinoma. These behavioural traits indicate the feasible identification of carcinoma cells in culture according to the proposed concept of the carcinoma cell dynamic phenotype. If further developed, this approach could later serve in a new functional online analysis of reactions of carcinoma cells to therapy. Such efforts conform to current trends in precision medicine.

• MeSH
• biopsie MeSH
• buněčný cyklus fyziologie   MeSH
• holografie metody   MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikroskopie metody   MeSH
• nádorové biomarkery metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• nádory hlavy a krku metabolismus   patologie   MeSH
• pohyb buněk fyziologie   MeSH
• senioři MeSH
• spinocelulární karcinom metabolismus   patologie   MeSH
• transkripční faktory metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.

• MeSH
• histologické techniky přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikroskopie přístrojové vybavení   metody   MeSH
• nervový systém cytologie   MeSH
• neurovědy MeSH
• savci MeSH
• zobrazování trojrozměrné metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

Thy-1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy-1 in mast cells and basophils induces activation events independent of the expression of Fcepsilon receptor I (FcepsilonRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy-1 and FcepsilonRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy-1.1 and exogenous Thy-1.2 genes and analyzed topography of the Thy-1 isoforms and Thy-1-induced signaling events. Light microscopy showed that both Thy-1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross-talk between Thy-1 isoforms and between Thy-1 and FcepsilonRI. This cross-talk was dependent on actin filaments. Thy-1 aggregates colocalized with two transmembrane adaptor proteins, non-T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy-1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI-anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aktiny metabolismus   MeSH
• antigeny Thy-1 genetika   metabolismus   MeSH
• financování organizované MeSH
• fosfoproteiny metabolismus   MeSH
• imunoblotting MeSH
• imunoelektronová mikroskopie MeSH
• interakce mezi receptory a ligandy MeSH
• krysa rodu rattus MeSH
• mastocyty imunologie   metabolismus   ultrastruktura   MeSH
• membránové mikrodomény ultrastruktura   MeSH
• membránové proteiny metabolismus   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• receptory IgE imunologie   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální transdukce imunologie   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antigeny CD9 fyziologie   MeSH
• antigeny CD98 - lehké řetězce metabolismus   MeSH
• antigeny metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• chemotaxe MeSH
• cytoskelet metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• glukuronidasa metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   MeSH
• krysa rodu rattus MeSH
• mastocyty cytologie   MeSH
• membránové proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• receptory IgE metabolismus   MeSH
• transportní systém aminokyselin y+ metabolismus   MeSH
• tyrosin chemie   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH