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.
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.
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
- Anesthesia MeSH
- Middle Cerebral Artery physiology MeSH
- Arterioles physiology MeSH
- Microscopy, Fluorescence methods MeSH
- Glymphatic System physiology MeSH
- Brain ultrastructure MeSH
- Cerebrospinal Fluid metabolism MeSH
- Cerebrovascular Circulation physiology MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Neuroimaging methods MeSH
- Sleep physiology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- Publication type
- Journal Article 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.
... The basic principles of the electron microscope 1 -- 1.1 The use of electrons for microscopy 2 -- 1.2 ... ... Electron diffraction 110 -- 3.10.2 High dispersion electron diffraction 112 -- 3.10.3 Dark field microscopy ... ... Processing plates - dish (tray) method 246 -- 7.5.3 Processing plates - tank method 247 -- 7.5.4 Processing sheet ... ... 7.9.1 The binocular viewing telescope 270 -- 7.9.2 The fluorescent screen 271 -- 7.9.3 The ambient lighting ... ... Energy analysis 316 -- 9.3.4 Scanning electron diffraction 319 -- 9.4 Scanning transmission electron microscopy ...
Practical methods in electron microscopy ; v. 2
Laboratory edition xii, 345 stran : ilustrace ; 23 cm
- MeSH
- Microscopy, Electron MeSH
- Physics MeSH
- Microscopy MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Fyzika
- NML Fields
- fyzika, biofyzika
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
- Histological Techniques instrumentation methods MeSH
- Humans MeSH
- Microscopy instrumentation methods MeSH
- Nervous System cytology MeSH
- Neurosciences MeSH
- Mammals MeSH
- Imaging, Three-Dimensional methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review 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
- Adaptor Proteins, Signal Transducing metabolism MeSH
- Actins metabolism MeSH
- Thy-1 Antigens genetics metabolism MeSH
- Financing, Organized MeSH
- Phosphoproteins metabolism MeSH
- Immunoblotting MeSH
- Microscopy, Immunoelectron MeSH
- Receptor Cross-Talk MeSH
- Rats MeSH
- Mast Cells immunology metabolism ultrastructure MeSH
- Membrane Microdomains ultrastructure MeSH
- Membrane Proteins metabolism MeSH
- Protein Isoforms genetics metabolism MeSH
- Receptors, IgE immunology metabolism MeSH
- Fluorescence Resonance Energy Transfer MeSH
- Signal Transduction immunology MeSH
- Transfection MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
Background and Aims: The actin cytoskeleton forms a dynamic network in plant cells. A single-point mutation in the DER1 (deformed root hairs1) locus located in the sequence of ACTIN2, a gene for major actin in vegetative tissues of Arabidopsis thaliana, leads to impaired root hair development (Ringli C, Baumberger N, Diet A, Frey B, Keller B. 2002. ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis. Plant Physiology129: 1464-1472). Only root hair phenotypes have been described so far in der1 mutants, but here we demonstrate obvious aberrations in the organization of the actin cytoskeleton and overall plant development. Methods: Organization of the actin cytoskeleton in epidermal cells of cotyledons, hypocotyls and roots was studied qualitatively and quantitatively by live-cell imaging of transgenic lines carrying the GFP-FABD2 fusion protein and in fixed cells after phalloidin labelling. Patterns of root growth were characterized by FM4-64 vital staining, light-sheet microscopy imaging and microtubule immunolabelling. Plant phenotyping included analyses of germination, root growth and plant biomass. Key Results: Speed of germination, plant fresh weight and total leaf area were significantly reduced in the der1-3 mutant in comparison with the C24 wild-type. Actin filaments in root, hypocotyl and cotyledon epidermal cells of the der1-3 mutant were shorter, thinner and arranged in more random orientations, while actin bundles were shorter and had altered orientations. The wavy pattern of root growth in der1-3 mutant was connected with higher frequencies of shifted cell division planes (CDPs) in root cells, which was consistent with the shifted positioning of microtubule-based preprophase bands and phragmoplasts. The organization of cortical microtubules in the root cells of the der1-3 mutant, however, was not altered. Conclusions: Root growth rate of the der1-3 mutant is not reduced, but changes in the actin cytoskeleton organization can induce a wavy root growth pattern through deregulation of CDP orientation. The results suggest that the der1-3 mutation in the ACT2 gene does not influence solely root hair formation process, but also has more general effects on the actin cytoskeleton, plant growth and development.
... from chicken eggs, 66 -- 3.4 Preparation of fragments of IgG, 68 -- 3.4.1 Separation of heavy and light ... ... - 3.5 Isolation of IgM, 79 -- 3.6 Preparation of fragments of IgM, 83 . 3.6.1 Separation of heavy, light ... ... from milk and colostrum, 91 -- 3.8 Preparation of fragments of IgA, 92 -- 3.8.1 Separation of heavy, light ... ... , 322 -- 13.2.6 Labelling tissue sections for immunofluorescence microscopy, 326 -- 13.3 Enzyme label ... ... - 13.3.4 Alkaline phosphatase-anti-alkaline phosphatase (APAAP) labelling, 337 -- 13.4 Electron microscopy ...
3rd ed. 362 s. : obr.
- Keywords
- Chemie, Imunita,
- MeSH
- Immunochemistry MeSH
- Immunologic Techniques MeSH
- Publication type
- Laboratory Manual MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- alergologie a imunologie
