Gli autori desiderano ringraziare il Prof. Nicola Smirnoff dell&#39;Università di Exeter per i suoi consigli e l&#39;Università di meccanismo di Bioimmagini Exeter. Il finanziamento è stato fornito dal Regno Unito Biotecnologie e Scienze Biologiche Research Council (sovvenzione di riferimento BB/E002682/1).

Il protocollo indicati di seguito descrive un metodo semplice per l&#39;utilizzo di PFD come mezzo infiltrativa di montaggio in foglie di Arabidopsis thaliana, ma possiamo anticipare che questo metodo può essere utilizzato in una varietà di applicazioni di imaging in cui si desidera l&#39;aria ricca di tessuti. 1. Campioni di foglie di montaggio in PFD <ol><li> Preparare un vetrino da microscopio con un gas-permeabili guarnizione di polidimetilsilossano (PDMS, Carolina Gel di osservazione). PDMS è un polimero viscoelastico e può essere modellata per fornire una camera di misura per esigenze sperimentali. </li><li> Equilibrare PFD con l&#39;aria. Ciò può essere ottenuto facendo gorgogliare aria o agitando un piccolo volume di PFD in un aria piena di bottiglia. </li><li> Decantare PFD in un piatto aperto Petri e galleggiare una piantina intera o foglia asportato il PFD per 5 minuti. Il tessuto deve diventare trasparente, che ricorda di tessuto vetrificato (Figura 2 (a)). Le foglie possono apparire più scura o più chiara rispetto a prima esposizione a PFD, dipendenzading in condizioni di illuminazione e l&#39;età dei tessuti utilizzati. </li><li> Riempire la camera di PDMS di aria equilibrato PFD e con attenzione trasferire i campioni di tessuto dal piatto di incubazione Petri alla camera di PDMS. Sigillare il vetrino con un coprioggetto e l&#39;immagine a seconda delle esigenze sperimentali. </li><li> Nota: PFD funzioni anche bene in una camera aperta costruita su un vetrino o in una camera di perfusione ed è compatibile con grasso al silicone. PFD non è compatibile con i componenti in Teflon, come li dissolve. </li></ol> 2. Rappresentante dei risultati: Ispezione al microscopio di PFD-incubate campioni di foglie dimostra che la maggior parte di spazi aerei sono inondate. Figura 2 (b) mostra spazi aerei inondato di ricombinanti GFP sospeso in PFD. E &#39;evidente che la foglia è invaso su incubazione con PFD, ma che occasionalmente sacche d&#39;aria può rimanere. L&#39;acqua non inondare la foglia in queste condizioni. LSCM dei campioni incubati e montato in una<html> ir, acqua o spettacoli PFD PFD che dà un vantaggio per l&#39;acqua e l&#39;aria nella profondità delle immagini possibili (Figura 3). L&#39;esempio riportato in Figura 3 mostra Arabidopsis aria, acqua e PFD montato lascia esprimere cytosolically localizzato Venere, una variante di YFP 11 che si esprime costitutivamente sotto il promotore 35S. Possiamo vedere di circa 2 volte maggiore profondità delle immagini quando si confrontano PFD e acqua. Va notato, tuttavia, che il miglioramento preciso visto quando si usa PFD varia con apertura numerica della lente e il tipo di tessuto utilizzato. Abbiamo visto in streaming citoplasmatici e capelli allungamento radice in PFD piantine trattate, ognuno indicativo di piante sane. Inoltre abbiamo dimostrato che 2 Fv / Fm, una misura del funzionamento di fotosintesi delle piante 12 rimane entro limiti tollerabili su scale temporali utilizzati nella diagnostica per immagini. 3394fig1.jpg &quot;/&gt; Figura 1. Impianto di complessità e le implicazioni foglia ottica Diagrammatical rappresentazione che mostra le caratteristiche anatomiche della A. foglia thaliana in relazione al set-up ottico. Abbreviazioni usate sono obj. = Lente dell&#39;obiettivo, imm. = Fluido immersione, cov. = Coprioggetti, mnt. = Montaggio, taglio. = Cuticola, annuncio. ep. = Epidermide adaxial, st. = Poro stomatica, sp. = Mesofillo spugnoso, come = spazio aereo, amico. = Palizzata mesofillo, vb = fascio vascolare, annuncio. ep. = Epidermide adaxial. Pareti cellulari sono indicati da linee nere. <img alt="Figura 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Figura 2. PFD penetra facilmente foglie di Arabidopsis (A) Foglie incubate in aria, acqua o PFD per 5 minuti e ripreso con una fotocamera digitale Leica DCF3000FX con un microscopio Leica MZ16F (Leica Microsystems (UK) Ltd., Milton-Keynes, UK). Il logo Giove era stampato su pellicola di acetato ed illuminato indietrom al di sotto con una scatola di luce. Tempo di esposizione è stato di 89 ms e tutte le immagini sono stati raccolti e trattati in modo identico. Barre di scala rappresentano 2 mm. (B) PFD portando ricombinante purificata proteina verde fluorescente (GFP), delimita spazi aerei in vivo. GFP è falso colore verde e rosso è usato per mostrare l&#39;auto-fluorescenza della clorofilla, che delimita le cellule del mesofillo. (Figura 2 (b) riprodotto con il permesso e tutti i dettagli tecnici disponibili in Littlejohn et al. 2010 2). Barre di scala rappresentano il 25 m. <img alt="Figura 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Figura 3. PFD migliora l&#39;imaging confocale in foglie Immagini che mostrano LSCM cytoplasmically localizzato Venere fluorescenza (verde) e autofluorescenza della clorofilla (rosso) in A. thaliana lascia intatto ripreso in aria, acqua o PFD. Lunghezza d&#39;onda di eccitazione è 514 nm ed emissione di fluorescenza è stato registrato a 518 nm 604 nm per Venere e at 657 nm679 nm per cloroplasti. Ogni pannello è un&#39;unica sezione confocale preso da una pila di Z-11 immagini acquisite confocale a intervalli di 5 micron. Questi sono stati estratti da un pieno Z-stack di 59 immagini acquisite ad intervalli di 1 micron, che sono stati usati per produrre film supplementari. Misure di profondità sono date relativamente alla superficie epidermica. Immagini rappresentative, che sono stati trattati in modo identico, vengono visualizzati. Dettagli sperimentali come le impostazioni microscopio sono identiche a quelle trovate in Littlejohn et al., 2010 2. Scalebars rappresentano il 20 micron.

<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>

Nessun conflitto di interessi dichiarati.

Questa è una tecnica semplice e facile da usare per migliorare la microscopia di aria riempita o otticamente complessi tessuti. Abbiamo dimostrato che la tecnica presenta alcuni vantaggi forte e speriamo che possa essere utilizzato per chiarire questioni biologiche riguardanti l&#39;aria ricca di tessuti. Per esempio sarebbe una scelta naturale per gli studi di attacco patogeno in mesofillo pianta o nei polmoni. Siamo anche consapevoli dei limiti della tecnica. Stiamo lavorando su una migliore corrispondenza indice di rifrazione, l&#39;uso con altri modi di microscopia, e la ricarica perfluorocarburo montaggio durante lunghi esperimenti tempi. Siamo anche consapevoli, però, che uno dei vantaggi principali di PFC, cioè di essere biologicamente inerte è uno rovescio della medaglia. PFC non si dissolvono rapidamente molecole biologiche che implica anche che non possono essere facilmente usati per fornire composti di interesse come gli ormoni, farmaci e altre piccole molecole o ioni.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Perfluorodecalin </td><td> F2 chimici </td><td> N / A </td><td> Telefono per ordinare. </td></tr><tr><td> Carolina di osservazione Gel </td><td> Lame biologica </td><td> 13-2700 </td><td></td></tr></tbody></table>

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

Il problema di acquisizione di immagini ad alta risoluzione in profondità in campioni biologici è ampiamente riconosciuto 1. In aria piena di tessuti come il mesofillo spugnoso di foglie delle piante o dei polmoni dei vertebrati ulteriori difficoltà derivano da transizioni multiple in indice di rifrazione tra i componenti cellulari, tra le celle e spazi aerei e tra il tessuto biologico e il resto del sistema ottico. Inoltre, discordanze indice di rifrazione portare ad attenuazione di eccitazione fluoroforo ed emissione del segnale in microscopia a fluorescenza. Descriviamo qui l&#39;applicazione della perfluorocarburo, perfluorodecalin (PFD), come mezzo di imaging ottico infiltrativa che migliora la microscopia confocale a scansione laser (LSCM) immagini campione in profondità, senza ricorrere ad aumenti di danneggiare in potenza del laser e ha un minimo impatto fisiologico 2. Noi descriviamo il protocollo per l&#39;utilizzo del PFD con il tessuto foglia Arabidopsis thaliana, che è otticamente più complesse a causa della suastruttura (Figura 1). PFD ha una serie di attributi che lo rendono adatto per questo uso 3. L&#39;indice di rifrazione di PFD (1,313) è paragonabile a quella dell&#39;acqua (1.333) ed è più vicina a quella del citosol (circa 1,4) rispetto all&#39;aria (1.000). Inoltre, PFD è prontamente disponibile, non fluorescente e non è tossico. La bassa tensione superficiale di PFD (19 dyne cm -1) è inferiore a quella dell&#39;acqua (72 dyne cm -1) e anche al di sotto del limite (25 - 30 dyne cm -1) per la penetrazione stomatica 4, che permette di inondare gli spazi aerei mesofillo spugnoso senza l&#39;applicazione di un vuoto potenzialmente distruttivi o tensioattivo. Infine e soprattutto, PFD ha una grande capacità di dissoluzione di CO 2 ed O 2, che consente lo scambio di gas deve essere mantenuta nel tessuto invaso, minimizzando così l&#39;impatto fisiologico sul campione. Queste proprietà sono state utilizzate in varie applicazioni che includono parziale respiro liquido e del polmone inflazionezione 5,6, chirurgia 7, sangue artificiale 8, ossigenazione della crescita dei media 9, e gli studi di formazione di cristalli di ghiaccio nelle piante 10. Attualmente, è comune a montare il tessuto in acqua o in tampone acquoso per vivere l&#39;imaging confocale. Riteniamo che l&#39;uso di PFD come un mezzo di montaggio rappresenta un miglioramento rispetto alla pratica esistente e consente la semplice preparazione di live campioni foglia intera per l&#39;imaging.

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

Descriviamo l&#39;uso di perfluorodecalin come mezzo infiltrante di montaggio. Questo è un metodo semplice per migliorare la profondità delle immagini in<em&gt; Arabidopsis thaliana</em&gt; Tessuto di foglie con il minimo impatto fisiologico.

Un metodo semplice per l&#39;imaging Arabidopsis Foglie Utilizzando Perfluorodecalin come Media Imaging infiltrativa

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 Views.  The University of Exeter. Descriviamo l'uso di perfluorodecalin come mezzo infiltrante di montaggio. Questo è un metodo semplice per migliorare la profondità delle immagini in Arabidopsis thaliana Tessuto di foglie con il minimo impatto fisiologico.

Video: 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.

Pirosequenziamento: un metodo semplice per genotipizzazione accurata

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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Biologia

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.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

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.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza 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.

Impostazione di un semplice foglio microscopio ottico per<em&gt; In Toto</em&gt; Imaging di<em&gt; C. elegans</em&gt; Sviluppo

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 tecnica infiltrazione-centrifugazione per l&#39;estrazione di apoplastico fluido da foglie delle piante Utilizzo<em&gt; Phaseolus vulgaris</em&gt; Come un esempio

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 in idrogel tridimensionali

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.

Discriminazione e caratterizzazione delle popolazioni eterocellulari con tecniche di imaging 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.

Metodo standardizzato per la sterilizzazione di High-throughput di semi di 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.

Analisi di Imaging di complementazione di luciferase in foglie di Nicotiana benthamiana per transitoriamente determinare dinamiche di interazione proteina-proteina

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.

FITC-Destrano di imaging come Reporter per esocitosi regolata

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