Die Autoren bedanken sich bei Prof. Nicholas Smirnoff von der University of Exeter für seine Beratung und der University of Exeter Bioimaging Einrichtung bedanken. Die Finanzierung wurde von der britischen Biotechnology and Biological Sciences Research Council (Stipendium Referenz BB/E002682/1) zur Verfügung gestellt.

Das Protokoll gegeben unten beschreibt eine einfache Methode für die Verwendung von PFD als infiltrative Eindeckmedium in Arabidopsis thaliana Blätter, aber wir rechnen damit, dass diese Methode in einer Vielzahl von Anwendungen, bei denen bildgebende Luft-reichen Gewebe gewünscht wird, verwendet werden. 1. Montage Blattproben in PFD <ol><li> Bereiten Sie einen Objektträger mit einer gasdurchlässigen Dichtung aus Polydimethylsiloxan (PDMS, Carolina Observation Gel). PDMS ist ein viskoelastisches Polymer und kann geformt, um eine Kammer zugeschnitten auf experimentellen Anforderungen zu liefern. </li><li> Äquilibrieren PFD mit Luft. Dies kann durch Einblasen von Luft oder durch Schütteln einem kleinen Volumen in einen mit Luft gefüllten Flasche PFD erreicht werden. </li><li> Dekantieren PFD in eine offene Petrischale und Schwimmer eine ganze Keimling oder herausgeschnitten Blatt auf PFD für 5 Minuten. Das Gewebe sollte durchscheinend, erinnert an verglasten Gewebe (Abbildung 2 (a)). Blätter erscheinen dunkler oder heller als vor der Belichtung auf PFD, abhängigding auf den Lichtverhältnissen und dem Alter des Gewebes verwendet werden. </li><li> Füllen Sie das PDMS Kammer mit Luft-äquilibriert PFD und sorgfältig Übertragung der Gewebeproben aus der Inkubation Petrischale auf die PDMS Kammer. Verschließen Sie die Folie mit einem Deckglas und Bild nach experimentellen Anforderungen. </li><li> Hinweis: PFD auch gut in eine offene Kammer auf einem Deckglas gebaut oder in einer Perfusionskammer und ist kompatibel mit Silikonfett. PFD ist nicht kompatibel mit Teflon-Komponenten, wie es sie löst. </li></ol> 2. Repräsentative Ergebnisse: Eine mikroskopische Untersuchung der PFD-inkubiert Blattproben zeigt, dass die Mehrheit der Lufträume überschwemmt werden. Abbildung 2 (b) zeigt Lufträume mit rekombinantem GFP in PFD ausgesetzt überflutet. Es ist offensichtlich, dass das Blatt nach Inkubation mit PFD ist überschwemmt, aber das gelegentliche Lufteinschlüsse bleiben kann. Wasser kennt keine Flut das Blatt unter diesen Bedingungen. LSCM von Proben inkubiert und montiert in einem<html> ir-, Wasser-oder PFD zeigt, dass PFD einen Vorteil gegenüber Wasser und Luft in die Tiefe der Bildgebung möglich (Abbildung 3) gibt. Das Beispiel in Abbildung 3 angegeben zeigt Luft-, Wasser-und PFD montiert Arabidopsis Blättern ausdrücken cytosolically lokalisiert Venus, eine Variante des YFP 11, konstitutiv ist unter den 35S-Promotor exprimiert. Wir können sehen, einer ungefähr 2-fachen Anstieg in der Tiefe der Bildgebung beim Vergleich PFD und Wasser. Es sollte jedoch beachtet werden, dass die genaue Verbesserung bei der Verwendung von PFD mit numerischen Apertur des Objektivs und der Art des Gewebes verwendet wird, variiert. Wir haben Plasmaströmung und Wurzel Haar Dehnung in PFD behandelt Sämlinge, jedes für gesunde Pflanzen zu sehen. Darüber hinaus haben wir 2 gezeigt, dass Fv / Fm, ein Maß für die Photosynthese Funktionieren der Pflanzen 12 in tolerierbaren Grenzen über Zeitskalen in der Bildgebung verwendet wird, bleibt. 3394fig1.jpg &quot;/&gt; Abbildung 1. Pflanze Blatt Komplexität und optischen Auswirkungen Schematische Darstellung, die den anatomischen Merkmalen der A. thaliana Blatt in Bezug auf die optischen Aufbau. Verwendete Abkürzungen sind obj. = Objektiv, imm. = Immersionsflüssigkeit, cov. = Deckglas, mnt. = Deckfluessigkeit, geschnitten. = Kutikula, ad. ep. = Adaxial Epidermis, st. = Stomata Pore, sp. = Schwammige Mesophyll, as = Luftraum, Kumpel. = Palisade Mesophyll, vb = Leitbündel, ad. ep. = Adaxial Epidermis. Die Zellwände werden durch schwarze Linien angedeutet. <img alt="Abbildung 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Abbildung 2. PFD durchdringt leicht Arabidopsis Blättern (A) Blätter in Luft, Wasser inkubiert oder PFD für 5 Minuten und aufgenommen mit einer Leica DCF3000FX Digitalkamera mit einem Leica-Mikroskop MZ16F (Leica Microsystems (UK) Ltd, Milton-Keynes, UK). Die JoVE Logo wurde auf Acetat-Folie gedruckt und beleuchtete herm unterhalb mit einem leichten Box. Die Belichtungszeit betrug 89 ms und alle Bilder wurden gesammelt und verarbeitet identisch. Scale-Balken 2 mm. (B) PFD tragen gereinigten rekombinanten grün fluoreszierende Protein (GFP) begrenzt Lufträume in vivo. GFP ist falsch grün und rot wird verwendet, um Chlorophyll Autofluoreszenz, welche die Mesophyllzellen begrenzt zeigen. (Abbildung 2 (b) mit Genehmigung und voller technischer Details in Littlejohn et al. 2010 2). Scale-Balken repräsentieren 25 m. <img alt="Abbildung 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Abbildung 3. PFD verbessert konfokale Bildgebung in den Blättern LSCM Bilder zeigen zytoplasmatisch lokalisiert Venus-Fluoreszenz (grün) und Chlorophyll-Autofluoreszenz (rot) in intakten A. thaliana Blätter in Luft, Wasser oder PFD abgebildet. Die Anregungswellenlänge betrug 514 nm und Fluoreszenz-Emission wurde bei 518 nm aufgezeichnet 604 nm für Venus und 657 nm679 nm für die Chloroplasten. Jedes Panel ist eine einzige konfokalen Ausschnitt aus einer Z-Stapel von 11 konfokalen Bildern bei 5 um Abständen erworben genommen. Diese wurden von einer vollständigen Z-Stapel von 59 Bildern bei 1 um Intervalle, die verwendet wurden, um ergänzende Filme zu produzieren erworben extrahiert. Tiefe Messungen sind relativ zu den epidermalen Oberfläche. Repräsentative Bilder, die identisch verarbeitet wurden, werden angezeigt. Experimentelle Details wie Mikroskop-Einstellungen sind identisch mit denen in Littlejohn et al., 2010 2 gefunden. Scalebars repräsentieren 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>

Keine Interessenskonflikte erklärt.

Dies ist eine einfache und leicht zu bedienen Technik Mikroskopie von luftgefüllten oder optisch komplexen Geweben zu verbessern. Wir haben gezeigt, dass die Technik einige starke Vorteile hat und wir hoffen, dass es verwendet wird, um biologische Fragen zu Luft-reichen Geweben aufzuklären. Zum Beispiel wäre es eine natürliche Wahl für die Untersuchung der Erreger Angriff in Anlage Mesophyll oder in der Lunge sein. Wir sind uns der Grenzen der Technik. Wir sind auf eine bessere Brechungsindexanpassung arbeiten, mit anderen Formen der Mikroskopie und Perfluorcarbon Deckfluessigkeit Nachschub während der langen Zeitskala Experimente verwenden. Wir erkennen auch an, aber, dass einer der Hauptvorteile von FKW, nämlich als biologisch inert hat eine Kehrseite. PFCs nicht gut lösen biologische Moleküle, die auch bedeutet, dass sie nicht einfach verwendet werden, um Verbindungen von Interesse wie Hormone, Medikamente und andere kleine Moleküle oder Ionen zu liefern.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Perfluordecalin </td><td> F2 Chemicals </td><td> N / A </td><td> Telefon zu bestellen. </td></tr><tr><td> Carolina Observation Gel </td><td> Blades Biological </td><td> 13-2700 </td><td></td></tr></tbody></table>

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

Das Problem des Erwerbs von hochauflösenden Bildern tief in biologischen Proben wird allgemein 1 quittiert. In luftgefüllte Gewebe wie die schwammige Mesophyll der Blätter der Pflanze oder Wirbeltier-Lungen weitere Schwierigkeiten ergeben sich aus mehrere Übergänge im Brechungsindex zwischen zellulären Komponenten, zwischen Zellen und Lufträume und zwischen dem biologischen Gewebe und der Rest des optischen Systems. Darüber hinaus führen Brechungsindex Missverhältnis zur Dämpfung von Fluorophor Anregungs-und Emissions-Signal in der Fluoreszenzmikroskopie. Wir beschreiben hier die Anwendung der Perfluorcarbon, Perfluordecalin (PFD), als infiltrative Imaging Medium, das optisch verbessert konfokaler (LSCM) Probenabbildung in der Tiefe, ohne auf schädliche Anstieg der Laserleistung und hat nur minimale physiologische Wirkung 2. Wir beschreiben das Protokoll für den Einsatz von PFD mit Arabidopsis thaliana Blattgewebe, die optisch komplexer als Ergebnis seiner istStruktur (Abbildung 1). PFD hat eine Reihe von Attributen, die es für diese Verwendung geeignet 3 machen. Der Brechungsindex von PFD (1.313) ist vergleichbar mit der des Wassers (1,333) und ist näher an der Cytosol (ca. 1,4) als Luft (1,000). Darüber hinaus ist PFD leicht verfügbar, nicht-fluoreszierenden und ist ungiftig. Die geringe Oberflächenspannung des PFD (19 Dyn cm -1) ist niedriger als die von Wasser (72 Dyn cm -1) und auch unter dem Grenzwert (25 bis 30 Dyn cm -1) für Stomata Eindringen 4, die es zu Überschwemmungen erlaubt das schwammige Mesophyll Lufträumen, ohne die Anwendung eines potenziell destruktiven Vakuum oder Tensid. Schließlich und vor allem hat PFD eine große Kapazität zur Auflösung von CO 2 und O 2, die den Gasaustausch in den überfluteten Gewebe aufrechterhalten werden, damit die Minimierung der physiologischen Wirkung auf die Probe ermöglicht. Diese Eigenschaften wurden in verschiedenen Anwendungen, die teilweise flüssige Atmung und Lunge Inflation sind benutzt wordenTION 5,6, Chirurgie 7, künstlichem Blut 8, Sauerstoffversorgung des Wachstums Media 9, sowie Studien über die Bildung von Eiskristallen in Pflanzen 10. Derzeit ist es üblich, Gewebe in Wasser oder wässrigem Puffer für Live-konfokale Abbildung montieren. Wir denken, dass die Verwendung von als Eindeckmedium PFD eine Verbesserung gegenüber der bisherigen Praxis dar und ermöglicht die einfache Herstellung von Live ganzen Blattproben für die Bildgebung.

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

Wir beschreiben die Verwendung von Perfluordecalin als infiltrative Eindeckmedium. Dies ist eine einfache Methode zur Verbesserung der Tiefe der Bildgebung in<em&gt; Arabidopsis thaliana</em&gt; Blattgewebe mit minimalen physiologischen Auswirkungen.

Eine einfache Methode zur Bildgebung Arabidopsis Leaves Mit Perfluordecalin als Infiltrative Imaging Medium

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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Research

JoVE Journal

Biology

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

21.5K Aufrufe.  The University of Exeter. Wir beschreiben die Verwendung von Perfluordecalin als infiltrative Eindeckmedium. Dies ist eine einfache Methode zur Verbesserung der Tiefe der Bildgebung in Arabidopsis thaliana Blattgewebe mit minimalen physiologischen Auswirkungen.

Serie: 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.

Pyrosequencing: Eine einfache Methode für genaue Genotypisierung

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

Forschung

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.

Ein Ex-ovo Chicken Embryo Culture-System Geeignet für Imaging und Mikrochirurgie Anwendungen

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.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit 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 ...

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.

Einrichten eines Simple Light Noten Mikroskop für<em&gt; In Toto</em&gt; Imaging von<em&gt; C. elegans</em&gt; Entwicklung

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.

Die Infiltration-Zentrifugation Technik zur Gewinnung von apoplastischen Flüssigkeit aus Pflanzenblättern verwenden<em&gt; Phaseolus vulgaris</em&gt; Als Beispiel

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 im dreidimensionalen Hydrogelen

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.

Diskriminierung und Charakterisierung von Heterocellular Populationen mit quantitativen Imaging-Techniken

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.

Standardisierte Methode für Hochdurchsatz-Sterilisation von Arabidopsis Samen

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.

Luciferase Komplementierung Imaging Assay in Nicotiana Benthamiana Blätter für Transient Bestimmung Proteinprotein Interaktion Dynamik

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.

Imaging-FITC-Dextran als Reporter für regulierte Exozytose

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