Les auteurs tiennent à remercier le professeur Nicholas Smirnoff de l&#39;Université d&#39;Exeter, pour ses conseils et de l&#39;Université de l&#39;installation de bio-imagerie Exeter. Le financement a été fourni par la biotechnologie et sciences biologiques au Royaume-Uni Conseil de recherches (subvention de référence BB/E002682/1).

Le protocole donné ci-dessous décrit une méthode simple pour l&#39;utilisation de VFI comme un moyen de montage d&#39;infiltration dans des feuilles d&#39;Arabidopsis thaliana, mais nous prévoyons que cette méthode peut être utilisée dans une variété d&#39;applications où l&#39;imagerie aérienne tissus riches est souhaitée. 1. Échantillons de feuilles de montage dans un VFI <ol><li> Préparer une lame de microscope avec un joint perméable aux gaz de polydiméthylsiloxane (PDMS, Caroline observation Gel). PDMS est un polymère viscoélastique et peut être moulé pour fournir une chambre adaptée aux besoins expérimentaux. </li><li> Equilibrer VFI avec l&#39;air. Ceci peut être réalisé par barbotage à l&#39;air ou en le secouant un petit volume de VFI dans une bouteille remplie d&#39;air. </li><li> Décanter PFD dans un plat de Petri ouverte et flottent un semis tout ou feuilles excisées sur le VFI pendant 5 minutes. Le tissu doit devenir translucide, rappelant des tissus vitrifiés (figure 2 (a)). Les feuilles peuvent apparaître plus foncées ou plus légers que avant l&#39;exposition au VFI, dépendanceDing sur les conditions d&#39;éclairage et de l&#39;âge du tissu utilisé. </li><li> Remplir la chambre de PDMS avec air-équilibrée PFD et transférer soigneusement les échantillons de tissus de l&#39;antenne d&#39;incubation de Petri à la chambre de PDMS. Sceau de la diapositive avec une lamelle et l&#39;image en fonction des besoins expérimentaux. </li><li> Remarque: PFD effectue aussi bien dans une chambre ouverte construite sur une lamelle ou dans une chambre de perfusion et est compatible avec de la graisse silicone. VFI n&#39;est pas compatible avec les composants en téflon comme il les dissout. </li></ol> 2. Les résultats représentatifs: L&#39;inspection microscopique des échantillons de feuilles VFI-incubées montre que la majorité des espaces aériens sont inondées. La figure 2 (b) montre espaces inondés recombinante GFP en suspension dans un VFI. Il est évident que la feuille est inondé par incubation avec un VFI, mais que des poches occasionnelles d&#39;air peuvent subsister. L&#39;eau n&#39;inonde pas les feuilles dans ces conditions. LSCM des échantillons incubés et monté dans un<html> ir, l&#39;eau ou VFI VFI montre donne un avantage sur l&#39;eau et l&#39;air dans la profondeur de l&#39;imagerie possible (figure 3). L&#39;exemple donné dans la figure 3 montre Arabidopsis air, l&#39;eau et des VFI montés laisse exprimer cytosolically localisée Vénus, une variante de la YFP 11 qui est exprimé de façon constitutive sous le promoteur 35S. Nous pouvons voir une multiplication par 2 environ augmentation de la profondeur de l&#39;imagerie en comparant VFI et de l&#39;eau. Il faut noter, cependant, que l&#39;amélioration précise vu lors de l&#39;utilisation VFI varie avec l&#39;ouverture numérique de la lentille et le type de tissu utilisé. Nous avons vu le streaming cytoplasmique et l&#39;allongement des poils racinaires de plants traités VFI, chaque indicatif de plantes saines. En outre, nous avons montré que les deux Fv / Fm, une mesure du fonctionnement photosynthétique des plantes 12 reste dans des limites tolérables sur des périodes utilisées dans l&#39;imagerie. 3394fig1.jpg &quot;/&gt; Figure 1. La complexité des feuilles des végétaux et des implications optiques Représentation schématique montrant les caractéristiques anatomiques de l&#39;A. feuilles thaliana par rapport à l&#39;optique de mettre en place. Les abréviations utilisées sont obj. = Lentille d&#39;objectif, imm. Liquide d&#39;immersion =, couv. = Lamelle, mnt. = Milieu de montage, couper. = Cuticule, ad. ep. = Épiderme adaxiale, st. = Pores stomatiques, sp. = Mésophylle spongieux, comme = espace aérien, mon pote. = Mésophylle palissade, vb = faisceau vasculaire, ad. ep. L&#39;épiderme = adaxiale. Les parois cellulaires sont indiquées par des lignes noires. <img alt="Figure 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Figure 2. VFI pénètre facilement des feuilles d&#39;Arabidopsis (A) Feuilles incubés dans l&#39;air, l&#39;eau ou un VFI pendant 5 minutes et imagée en utilisant un Leica DCF3000FX appareil photo numérique avec un microscope Leica MZ16F (Leica Microsystems (RU) Ltd, Milton-Keynes, Royaume-Uni). Le logo a été imprimé sur JoVE film d&#39;acétate et illuminé BOFm au-dessous d&#39;une boîte à lumière. Le temps d&#39;exposition était de 89 ms et toutes les images ont été collectées et traitées de manière identique. Barres d&#39;échelle représentent de 2 mm. (B) VFI portant recombinant purifié la protéine fluorescente verte (GFP) délimite espaces aériens in vivo. GFP est faux de couleur verte et rouge est utilisé pour montrer l&#39;auto-fluorescence chlorophyllienne, qui délimite les cellules du mésophylle. (Figure 2 (b) reproduit avec la permission et plein de détails techniques disponibles dans Littlejohn et al. 2010 2). Barres d&#39;échelle représentent 25 m. <img alt="Figure 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Figure 3. VFI améliore l&#39;imagerie confocale dans les feuilles Images montrant LSCM cytoplasme localisée Venus de fluorescence (vert) et autofluorescence chlorophylle (rouge) chez A. thaliana intacte feuilles imagé dans l&#39;air, l&#39;eau ou de VFI. Longueur d&#39;onde d&#39;excitation était de 514 nm et d&#39;émission de fluorescence a été enregistré à 518 nm 604 nm pour Vénus et 657 nm au679 nm pour les chloroplastes. Chaque panneau est une seule section confocale prises à partir d&#39;un Z-stack de 11 images acquises confocale à intervalles de 5 um. Ils ont été extraites d&#39;une pleine Z-stack de 59 images acquises à intervalles de 1 um, qui ont été utilisés pour produire des films supplémentaires. Les mesures de profondeur sont donnés par rapport à la surface de l&#39;épiderme. Images représentant, qui ont été traitées de façon identique, sont affichées. Les détails expérimentaux tels que les paramètres de microscope sont identiques à ceux trouvés dans Littlejohn et al. 2010 2. Barres d&#39;échelle représentent 20 um.

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

C&#39;est une technique simple et facile à utiliser pour améliorer la microscopie remplis d&#39;air ou optiquement complexes tissus. Nous avons montré que la technique a des avantages solides et nous espérons qu&#39;il sera utilisé pour élucider des questions biologiques se rapportant à l&#39;air riche en tissus. Par exemple, il serait un choix naturel pour les études de l&#39;attaque de pathogènes dans mésophylle des plantes ou dans les poumons. Nous sommes également conscients des limites de la technique. Nous travaillons sur une meilleure adéquation entre l&#39;indice de réfraction, l&#39;utilisation des autres modes de microscopie et de réapprovisionnement perfluorocarbone milieu de montage pendant de longues expériences calendrier. Nous reconnaissons aussi, cependant, que l&#39;un des principaux avantages de PFC, à savoir être biologiquement inertes a une Flipside. PFC ne se dissolvent pas facilement des molécules biologiques qui implique également qu&#39;ils ne peuvent pas être facilement utilisé pour délivrer des composés d&#39;intérêt tels que les hormones, les médicaments et autres petites molécules ou d&#39;ions.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Perfluorodecalin </td><td> Produits chimiques F2 </td><td> N / A </td><td> Téléphone à l&#39;ordre. </td></tr><tr><td> Caroline observation Gel </td><td> Lames biologique </td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

Le problème de l&#39;acquisition d&#39;images haute résolution en profondeur dans des échantillons biologiques est largement reconnue 1. Dans l&#39;air rempli de tissus tels que le mésophylle spongieux des feuilles des plantes ou des poumons de vertébrés autres difficultés proviennent de plusieurs transitions de l&#39;indice de réfraction entre les composants cellulaires, entre les cellules et espaces aériens et entre les tissus biologiques et le reste du système optique. Par ailleurs, les inadéquations indice de réfraction conduire à une atténuation de l&#39;excitation et d&#39;émission du signal fluorophore en microscopie à fluorescence. Nous décrivons ici l&#39;application du perfluorocarbone, perfluorodécaline (VFI), comme un moyen d&#39;imagerie infiltrante qui améliore optiquement microscopie confocale à balayage laser (LSCM) de l&#39;échantillon d&#39;imagerie en profondeur, sans recourir à des augmentations d&#39;endommager la puissance du laser et a un impact minimal sur physiologiques 2. Nous décrivons le protocole d&#39;utilisation des VFI avec le tissu des feuilles d&#39;Arabidopsis thaliana, ce qui est optiquement complexes en raison de sesstructure (figure 1). PFD a un certain nombre d&#39;attributs qui le rendent approprié pour cet usage 3. L&#39;indice de réfraction du VFI (1,313) est comparable à celle de l&#39;eau (1,333) et est proche de celui de cytosol (environ 1,4) que l&#39;air (1.000). En outre, les VFI sont facilement accessibles, non fluorescentes et est non toxique. La faible tension de surface du VFI (19 dynes cm -1) est plus faible que celle de l&#39;eau (72 dynes cm -1) et également inférieur à la limite (25 - 30 cm Dyne -1) pour la pénétration stomatique 4, ce qui lui permet d&#39;inonder les espaces aériens mésophylle spongieux sans l&#39;application d&#39;un vide potentiellement destructeurs ou d&#39;un tensioactif. Enfin et surtout, VFI a une grande capacité à dissoudre le CO 2 et O 2, qui permet l&#39;échange de gaz pour être maintenu dans le tissu inondées, minimisant ainsi l&#39;impact physiologique sur l&#39;échantillon. Ces propriétés ont été utilisées dans différentes applications qui incluent la respiration partielle liquides et pulmonaires inflationtion 5,6, la chirurgie 7, 8 du sang artificiel, l&#39;oxygénation de la croissance des médias 9, et les études de la formation de cristaux de glace dans les plantes 10. Actuellement, il est courant de monter des tissus dans l&#39;eau ou tampon aqueux pour l&#39;imagerie confocale en direct. Nous considérons que l&#39;utilisation des VFI comme un milieu de montage représente une amélioration sur la pratique existante et permet la préparation simple de vivre ensemble des échantillons de feuilles pour l&#39;imagerie.

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Nous décrivons l&#39;utilisation de perfluorodécaline comme un moyen d&#39;infiltration de montage. C&#39;est une méthode simple pour améliorer la profondeur de l&#39;imagerie dans<em&gt; Arabidopsis thaliana</emTissus de la feuille&gt; avec impact physiologique minime.

Une méthode simple pour l&#39;imagerie Arabidopsis Feuilles utilisant perfluorodécaline comme un moyen d&#39;imagerie infiltratives

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K vues.  The University of Exeter. Nous décrivons l'utilisation de perfluorodécaline comme un moyen d'infiltration de montage. C'est une méthode simple pour améliorer la profondeur de l'imagerie dans Arabidopsis thaliana avec impact physiologique minime.

Vidéo: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyroséquençage: une méthode simple pour le génotypage précis

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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Biologie

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Une Ex-ovo- Poulet embryonnaire du système de culture approprié pour les applications d&#39;imagerie et de microchirurgie

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Analyse cellulaire spécifique de<em&gt; Arabidopsis</em&gt; Feuilles en utilisant la fluorescence par tri cellulaire activé

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

Configuration d&#39;une nappe de lumière Microscope simple pour<em&gt; En Toto</em&gt; Imagerie de<em&gt; C. elegans</em&gt; Développement

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La technique d&#39;infiltration-centrifugation pour l&#39;extraction de apoplastique Fluid feuilles des plantes Utilisation<em&gt; Phaseolus vulgaris</em&gt;, Un exemple

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET Imaging en hydrogels en trois dimensions

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Discrimination et caractérisation des populations hétéro-cellulaires à l&#39;aide de techniques d&#39;imagerie quantitative

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Méthode normalisée pour la stérilisation à haut débit des graines d’Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Analyse de l’imagerie de la complémentation de luciférase dans les feuilles de Nicotiana benthamiana pour transitoirement déterminant la dynamique Interaction protéine-protéine

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

L’imagerie FITC-dextran comme Reporter pour l’exocytose régulée

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...