Os autores gostariam de agradecer ao Prof Nicholas Smirnoff, da Universidade de Exeter para o seu conselho e da Universidade de Exeter Facility BioImaging. O financiamento foi fornecido pelo UK Biotecnologia e Ciências Biológicas Research Council (concessão de referência BB/E002682/1).

O protocolo a seguir descreve um método simples para usar PFD como um meio de montagem infiltrativa em folhas de Arabidopsis thaliana, mas prevemos que esse método pode ser usado em uma variedade de aplicações onde a imagem do ar rico em tecidos é desejada. 1. Montagem de amostras de folhas em PFD <ol><li> Prepare uma lâmina de microscópio com uma junta de gás-permeáveis ​​de polidimetilsiloxano (PDMS, Carolina Gel de Observação). PDMS é um polímero viscoelástico e pode ser moldada para proporcionar uma câmara adaptada aos requisitos experimental. </li><li> Equilibrar PFD com o ar. Isto pode ser obtido por borbulhamento de ar ou agitando um pequeno volume de PFD em uma garrafa cheia de ar. </li><li> Decantar PFD em uma placa de Petri abertas e flutuar uma muda todo ou em folhas excisadas PFD por 5 minutos. O tecido deve tornar-se translúcida, lembrando de tecido vitrificado (Figura 2 (a)). Folhas podem aparecer mais escuro ou mais claro do que antes da exposição ao PFD, depending nas condições de iluminação ea idade do tecido utilizado. </li><li> Encher a câmara de PDMS com ar-equilibradas PFD e transferir cuidadosamente as amostras de tecido do prato de incubação de Petri para a câmara de PDMS. Selar o slide com uma lamela e imagem de acordo com exigências experimental. </li><li> Nota: PFD também funciona bem em uma câmara aberta construída sobre uma lamela ou em uma câmara de perfusão e é compatível com graxa de silicone. PFD não é compatível com os componentes Teflon, uma vez que dissolve-los. </li></ol> 2. Resultados representativos: Exame microscópico de amostras PFD-incubadas folha mostra que a maioria dos espaços aéreos são inundadas. Figura 2 (b) mostra espaços aéreos inundado com recombinante GFP suspenso no PFD. É evidente que a folha é inundado após incubação com PFD, mas que os bolsões ocasionais de ar pode permanecer. Água não inundar a folha sob essas condições. LSCM de amostras incubadas e montado em um<html> ri, água ou PFD mostra que PFD dá uma vantagem sobre a água eo ar na profundidade de imagem possível (Figura 3). O exemplo dado na Figura 3 mostra ar, água e PFD Arabidopsis montado deixa expressar Venus cytosolically localizada, uma variante do YFP 11, que é expressa constitutivamente sob o promotor 35S. Podemos ver um aumento de aproximadamente 2 vezes na profundidade de imagem ao comparar PFD e água. Deve-se notar, no entanto, que a melhoria precisa visto quando usando PFD varia com abertura numérica da lente e do tipo de tecido utilizado. Vimos de streaming citoplasmática e alongamento de cabelo raiz em PFD mudas tratadas, cada indicativo de plantas saudáveis. Além disso, temos mostrado que 2 Fv / Fm, uma medida do funcionamento da fotossíntese das plantas 12 permanece dentro de limites toleráveis ​​em escalas de tempo usado em imagiologia. 3394fig1.jpg &quot;/&gt; Figura 1. Complexidade e as implicações de plantas de folha óptica Representação diagramática mostrando as características anatômicas do A. folha thaliana em relação à ótica set-up. Abreviaturas utilizadas são obj. = Objetiva de imm. = Fluido de imersão, cov. = Lamela, mnt. = Montagem, corte. = Cutícula, ad. ep. = Epiderme adaxial, st. = Poro estomático, sp. = Mesófilo esponjoso, como = espaço aéreo, amigo. = Mesofilo paliçádico, vb = feixe vascular do anúncio,. ep. = Epiderme adaxial. Paredes celulares são indicados por linhas pretas. <img alt="Figura 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Figura 2. PFD penetra facilmente folhas de Arabidopsis (A) Folhas incubadas em ar, água ou PFD por 5 minutos e fotografada usando uma câmera digital Leica DCF3000FX com um microscópio MZ16F Leica (Leica Microsystems (UK) Ltd., Milton-Keynes, UK). O logotipo Jove era impresso em filme de acetato e iluminado from abaixo com uma caixa de luz. Tempo de exposição foi de 89 ms e todas as imagens foram coletadas e processadas de forma idêntica. Barras de escala representam 2 mm. (B) PFD carregando recombinantes purificadas proteína verde fluorescente (GFP) delimita espaços aéreos in vivo. GFP é falsa cor verde, e vermelho é usado para mostrar a clorofila auto-fluorescência, que delimita as células do mesofilo. (Figura 2 (b) reproduzida com a permissão e os detalhes técnicos completos disponíveis em Littlejohn et al. 2010 2). Barras de escala representam 25 m. <img alt="Figura 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Figura 3. PFD melhora de imagem confocal em folhas LSCM imagens mostrando cytoplasmically localizada Venus fluorescência (verde) e autofluorescência da clorofila (vermelho) em A. thaliana deixa intacta fotografada no ar, água ou PFD. Comprimento de onda de excitação de 514 nm e emissão de fluorescência foi registrada a 518 nm 604 nm para Vênus e em 657 nm679 nm para os cloroplastos. Cada painel é uma única seção confocal tirada de um Z-stack de 11 imagens confocal adquiridos em intervalos de 5 mM. Estes foram extraídos de um total Z-stack de 59 imagens adquiridas em intervalos de 1 M, que têm sido usados ​​para produzir filmes complementares. Medições de profundidade são dados relativos à superfície epidérmica. Imagens representativas, que foram processadas de forma idêntica, são mostrados. Detalhes experimentais, tais como definições de microscópio são idênticos aos encontrados no Littlejohn et al., 2010 2. Scalebars representam 20 mM.

<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>

Não há conflitos de interesse declarados.

Esta é uma técnica simples e fácil de usar para melhorar a microscopia de ar-cheios ou opticamente complexas tecidos. Nós mostramos que a técnica tem algumas vantagens forte e esperamos que ele será usado para elucidar questões biológicas relacionadas com ar-ricos tecidos. Por exemplo, seria uma escolha natural para estudos de ataque de patógenos no mesofilo de plantas ou nos pulmões. Nós também estamos cientes das limitações da técnica. Estamos trabalhando em uma melhor adequação do índice de refração, use com outros modos de microscopia, perfluorocarbono e reposição de montagem durante longos experimentos prazo. Também reconhecemos, no entanto, que uma das principais vantagens do PFC, ou seja, sendo biologicamente inerte tem um flipside. PFCs não são facilmente dissolver moléculas biológicas que também implica que eles não podem ser facilmente usados ​​para compostos de interesse, tais como hormônios, drogas e outras pequenas moléculas ou íons.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Perfluorodecalin </td><td> F2 Chemicals </td><td> N / A </td><td> Telefone para a ordem. </td></tr><tr><td> Carolina observação Gel </td><td> Lâminas Biológica </td><td> 13-2700 </td><td></td></tr></tbody></table>

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

O problema de aquisição de imagens de alta resolução em profundidade amostras biológicas é amplamente reconhecido 1. No ar cheio de tecidos como o mesofilo esponjoso de folhas das plantas ou dos pulmões dos vertebrados mais dificuldades surgem de várias transições no índice de refração entre os componentes celulares, entre as células e espaços aéreos e entre o tecido biológico eo resto do sistema óptico. Além disso, desencontros índice de refração levar a atenuação da excitação fluoróforo e emissão de sinal em microscopia de fluorescência. Descrevemos aqui a aplicação do perfluorocarbono, perfluorodecalin (PFD), como um meio de imagens infiltrativa que melhora a ótica de varredura a laser microscopia confocal (LSCM) imagem da amostra em profundidade, sem recorrer a aumentos prejudiciais na potência do laser e tem impacto fisiológico mínimo 2. Nós descrevemos o protocolo para utilização do PFD com tecido de Arabidopsis thaliana folha, que é opticamente complexas, como resultado de suaestrutura (Figura 1). PFD tem uma série de atributos que o tornam adequado para este uso 3. O índice de refração do PFD (1,313) é comparável com a de água (1.333) e é mais próximo ao do citosol (aprox. 1,4) do que o ar (1,000). Além disso, PFD está prontamente disponível, não-fluorescentes e não é tóxico. A baixa tensão superficial da PFD (19 dinas cm -1) é menor que a da água (72 dinas cm -1) e também abaixo do limite (25 - 30 cm dyne -1) para a penetração estomática 4, que permite a inundação o mesofilo esponjoso espaços aéreos sem a aplicação de um vácuo potencialmente destrutiva ou surfactante. Finalmente, e crucialmente, PFD tem uma grande capacidade para dissolver CO 2 e O 2, que permite a troca de gás para ser mantido no tecido inundadas, minimizando assim o impacto fisiológico sobre a amostra. Estas propriedades têm sido usados ​​em várias aplicações que incluem respiração líquida parcial e pulmão inflação 5,6, a cirurgia 7, 8 sangue artificial, a oxigenação de crescimento media 9, e os estudos de formação de cristais de gelo nas plantas 10. Atualmente, é comum para montar o tecido em água ou tampão aquoso para a imagem latente confocal ao vivo. Consideramos que o uso de PFD como um meio de montagem representa uma melhoria em prática existente e permite a preparação simples de viver amostras de folhas inteiras para imagem.

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

Nós descrevemos o uso de perfluorodecalin como um meio de montagem infiltrativa. Este é um método simples para melhorar a profundidade da imagem em<em&gt; Arabidopsis thaliana</em&gt; Tecido foliar com impacto fisiológico mínimo.

Um método simples para criação de imagens Arabidopsis Folhas Usando Perfluorodecalin como um meio de imagem infiltrativas

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 visualizações.  The University of Exeter. Nós descrevemos o uso de perfluorodecalin como um meio de montagem infiltrativa. Este é um método simples para melhorar a profundidade da imagem em Arabidopsis thaliana Tecido foliar com impacto fisiológico mínimo.

Vídeo: 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: Um método simples para genotipagem Accurate

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

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.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

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.

Análise de célula específico<em&gt; Arabidopsis</em&gt; Folhas utilizando ordenação células activadas por fluorescência

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.

Configurando um Microscópio folha de luz simples para<em&gt; Em Toto</em&gt; Imagem de<em&gt; C. elegans</em&gt; Desenvolvimento

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.

A técnica de infiltração-centrifugação para Extração de apoplásticas Fluid das folhas da planta Usando<em&gt; Phaseolus vulgaris</em&gt; Como exemplo

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 no hidrogéis tridimensionais

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.

Discriminação e Caracterização de Populações Heterocelulares Usando Técnicas de Imagem Quantitativa

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.

Método padronizado para a esterilização da elevado-produção de sementes de 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.

Luciferase complementação Imaging ensaio em folhas de Nicotiana benthamiana de transitoriamente determinação dinâmica da interação da proteína-proteína

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.

Imagem latente FITC-dextrano como repórter por exocitose regulada

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