We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1. Aequorina Based Ca2 + Imagem Usando o Sistema FAS <ol><li> Preparar mudas para imagens de luminescência. Esterilizar as sementes de plantas de Arabidopsis expressando a Aequorina com uma solução de lixívia a 10% contendo 0,01% de Triton-100. Semear as sementes estéreis sobre uma placa quadrada (10 x 10 cm quadrados placa de Petri com grade,) contendo força total MS (Murashige e Skoog Basal Mistura de sal), 1% de sacarose, e 1.2% de ágar. Colocar as placas verticalmente em uma câmara de crescimento, após a estratificação a 4 ° C durante 2 dias (Figura 1A). </li><li> Transferir as plantas para um filme. Coloque uma película adesiva (Figura 1B) na parte superior, de 7-10 dias de idade em crescimento mudas na placa. Com cuidado, empurre o filme à mão para garantir que as mudas aderir ao filme (Figura 1C). Descascar suavemente a película de modo que as plântulas permanece aderida à película (Figura 1D e 1E) </li><li> Incubar as mudas com cofator. Coloque a umdhered mudas para a placa quadrada (10 x 10 cm) contendo 15 ml de 2 ug / ml de h-CTZ (coelenterazina) em água. Incubar as plântulas, à temperatura ambiente durante 4 horas durante a noite (Figura 1F). </li><li> Prepare-se para imagens de luminescência. Pegue o filme fora da solução h-CTZ e cortá-la ao meio, formando duas partes. Coloque cada pedaço de filme com mudas de face para cima em duas placas diferentes. Deixe as placas no escuro por 5 min. </li><li> Adquirir imagens de luminescência. No escuro, colocar as duas placas ao lado do outro na fase do sistema de imagem de luminescência (Figura 1G). Adquirir imagens imediatamente após a adição de 20 ml de solução de estímulos para as placas simultaneamente. </li><li> Analisar imagens de luminescência. Escolha a mesma faixa de exibição para todas as imagens de luminescência. Cortar a região de interesse (ROI) e gerar as imagens como arquivos JPEG (Figura 2A). Alternativamente, exportar as imagens como arquivos de formato SPE e importá-los para oSoftware de análise de imagem ImageJ. Definir medidas para cálculo do valor médio de cinza de ROI. Selecione o mesmo tamanho da área ROI e medir os dados cinza e presentes médios como gráficos de barras (Figura 2B). </li></ol> 2. vivo celular confocal Ca2 + Imagem <ol><li> Preparar mudas para imagem confocal. Esterilizar sementes de plantas de Arabidopsis expressando case12 com solução de água sanitária a 10% contendo 0,01% de Triton. Semear as sementes estéreis em uma placa contendo sais MS força integral, 1% de sacarose e 1,2% de ágar. Colocar as placas verticalmente em uma câmara de crescimento, após a estratificação a 4 ° C durante 2 dias. </li><li> Câmaras de imagem Setup <ol><li> Monte uma câmara de imagem usando uma lâmina e lamela (Câmara A). Coloque um pedaço de algodão embebido de água para o meio da lâmina. Em seguida, transferir uma ou duas mudas de cinco dias de idade a partir da placa para o topo da água encharcada algodão. Cole pequenos pedaços de barro para cada canto de uma lamela, umd lugar lamela no topo de mudas de criar um espaço (câmara) entre a lamela e corrediça (Figura 3A, painel esquerdo). Ligar uma extremidade do tubo de polietileno (0,58 mm de diâmetro) para uma seringa de 1 ml e colocar a outra extremidade do tubo imediatamente adjacente à câmara. Segurar o tubo no lugar com fita adesiva (Figura 3A, painel da direita). </li><li> Monte uma câmara de imagem utilizando uma câmara de plexiglass (Câmara B). <ol><li> Espalhar uma camada fina de massa de silicone em torno de dois tubos de polietileno (0,58 mm de diâmetro) e pressionar os tubos revestidos para os canais em cada lado da câmara de plexiglass (Figura 3B painel da esquerda). Espalhar uma camada fina de massa de silicone sobre a superfície da câmara que contém as ranhuras do tubo e apertar uma lamela na graxa para vedar um lado da abertura da câmara (cortadas). </li><li> Transferir uma plântula cinco dias de idade a partir da placa para a câmara e colocar um pedaço de algodão embebido em água na parte superior of a muda. Espalhe massa de silicone sobre a outra superfície da câmara, e pressionar outra lamela sobre a selar. Ligar a extremidade de um dos tubos a uma seringa de 1 ml. Deixar a extremidade do outro tubo aberto (painel direito Figura 3B). </li></ol></li><li> Preparar uma câmara de imagem usando uma tampa de vidro câmaras (Câmara C). Esterilizar o vidro da tampa câmaras com etanol 70% e deixá-la sobre o capô até secar. Adicionar 0,6 ml ou 0,2 ml de força total meio MS contendo 1% de sacarose e 0,5% de phytagel em cada poço de uma câmara de dois poços ou de uma câmara de 8 poços (Figura 3C painel da direita), respectivamente. Esterilizar sementes e semear as sementes diretamente em uma tampa de vidro câmaras contendo uma fina camada de gel transparente e deixá-los crescer verticalmente, durante 5 dias (Figura 3C). </li></ol></li><li> Adquirir imagens usando um microscópio confocal. Para aplicar estímulos de estresse, tais como sal, frio ou peróxido, lentamente injete cerca de 200 mL de NaCl 150 mM, ice-cold água ou 1 mM de H 2 O 2 a solução para dentro da câmara (Secção A ou B), imediatamente antes da obtenção de imagens, ou adicionar cuidadosamente 500 ul ou 100 ul de solução de estímulo para o poço de uma câmara 2 ou 8 poços, respectivamente . Capturar imagens imediatamente após a aplicação da solução de estímulo usando uma Nikon A1R microscópio confocal de varredura a laser invertido com uma lente de imersão em água 20X (abertura numérica 0,75). Coletar uma série temporal de imagens em intervalos de 4 s com comprimentos de onda de excitação e emissão de 488 nm e 500-550 nm, respectivamente, e com uma resolução de pixel de 512 x 512. </li><li> Análise de Imagem Usando Nikon Elements, ROIs foram desenhadas ao redor de cada célula (ou área) de interesse. Intensidade total dentro de cada ROI foi medido ao longo do tempo usando a caixa de diálogo Tempo de medição (ImageJ poderia ser usado em vez disso). Total de medições de intensidade foram exportados e processados ​​em DataGraph. Ca2 + spike amplitude foi definida como a intensidade do pico de menos descansando intensidade, duração como the de tempo entre o início ea conclusão de uma espiga, eo período como o intervalo de tempo entre os picos de pico adjacentes de dois picos. teste t foi utilizado para comparar as médias. </li></ol>

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The authors have nothing to disclose.

Nós demonstramos um sistema FAS para a gravação do espaço-temporal de Ca2 + resposta de mudas de Arabidopsis. Este FAS Ca2 + sistema de gravação proporciona uma abordagem simples, sensível e robusto, que pode ser adaptado para medir a Ca2 dinâmica desencadeada por estímulos diversos, para além dos estímulos de stress abiótico, que são aqui apresentados. Usando este sistema, podemos facilmente comparar tecido- ou específicos de estímulos espaço-temporais Ca2 + d...

A célula de planta responde ao meio ambiente através de sinalização que coordena as acções de células. Um evento de sinalização precoce de células em resposta a estímulos do meio ambiente é um aumento transitório de Ca2 +. O padrão, ou a assinatura de um aumento transitório da concentração de Ca2 + livre é caracterizado pela sua amplitude, frequência e duração. Assinaturas Ca2 + espaço-temporais distintas regular diferentes atividades celulares 1. Estímulos específicos, como o calor, o frio, o sal, a seca, a luz, ou hormônios vegetais, pode ajustar a atividade espaço-temporal localizada à membrana canais de Ca2 + e / ou transportadores, resultando em específicos de Ca2 + assinaturas. Embora os transportadores de Ca2 + têm sido bem caracterizados, pouco é conhecido sobre as identidades molecular e as funções de canais de Ca 2 + em plantas 1. Telas genéticos para os mutantes com alteração de Ca2 + resposta ao estresse pode ser um estímulo eficaz aproxoach para identificar os componentes que compõem Ca2 + assinaturas. Ca2 sistemas de detecção com base recentemente vários Aequorina têm sido desenvolvidos que facilitam telas genéticos para componentes de sinalização de Ca2 + em resposta a ataque patogénico e stress abiótico 2-4. Aequorina foi usado pela primeira vez para detectar sinais de Ca2 + em plantas no início de 1990 5. Desde então, a Aequorina foi orientada para diferentes compartimentos celulares, tais como o 5 citoplasma, núcleo 6, 7 cloroplastos, tonoplast 8, 9 mitocôndrias, e estroma 10, bem como para diferentes tipos de células na raiz para monitorar célula específica de Ca 2 + sinais 11. Medições de Ca 2 + base de Aequorina revelar o espacial e temporal de Ca2 + resposta de uma população de células ao estresse estímulos. No entanto, na maioria dos casos, as respostas do Ca2 + de células individuais são unsynchronized no tecido respondendo 4. Por conseguinte, a Aequorina Ca2 + gravação não necessariamente comunicar o sinal de Ca2 + em células individuais. Nos últimos anos, a proteína fluorescente geneticamente codificado (FP) à base de Ca2 + de indicadores, tais como cameleon amarelo (YCS) 12 e 13 casos12 têm sido utilizados para estudar a sinalização de Ca2 + com alta resolução subcelular. YCS são fluorescência transferência de energia de ressonância (FRET) à base de Ca 2 + indicadores, contendo PCP e YFP variantes ligadas pela Ca2 + ligaï¿½o ao calmodulina proteínas e peptídeos de ligação à calmodulina M13. A calmodulina sofre uma alteração conformacional, uma vez que se liga a Ca 2 +, pondo assim PCP e YFP mais juntos, o que resulta num aumento da transferência de energia (reforçada FRET). O nível de FRET ao longo do tempo, calculada aproximadamente como a razão entre a intensidade do sinal YFP PCP, reflecte intracelular de Ca2 + dinâmica. Várias versões YC têm sido utilizados em plantas. YC3.6 era targeted para o citosol 14,15, núcleo 16, 17 mitocôndrias e membrana plasmática 18, e YC4.6 e D4ER foram direcionados para o ER 15,19, e D3cpv foi direcionada para os peroxissomos 20. Plantas transgênicas expressando YCS permitir a geração de imagens de células vivas de Ca2 + dinâmica em diferentes compartimentos celulares de diferentes tipos de células. Casos (presumivelmente Ca lcium se nsor) são proteínas simples permuta circular fluorescentes (cpFPs) abrigam uma calmodulina e peptídeos de ligação à calmodulina M13. Após a ligação ao Ca2 +, os casos sofrer alterações conformacionais, que conduz a um aumento da intensidade da fluorescência. A correlação entre a resposta da fluorescência do CASE e concentração de Ca2 + permite intracelular de Ca2 + dinâmica a ser medido quantitativamente. A variante case12 tem 12 vezes maior de fluorescência nas Ca2 + formas saturados. N. plantas benthiminana transitoriamente expressing case12 ou plantas de Arabidopsis expressando estavelmente Caso 12 foram usados ​​para estudar Ca 2 + sinalização na defesa e abióticos de estresse 4,21. Asynchronous espacial e temporal Ca2 oscilações nas células respondem ao ataque de patógenos, ou ao estresse desidratação foram revelados com base case12 Ca2 + imagem. Aqui, apresentamos instruções detalhadas para a Aequorina imagem luminescência base de Tecido e estímulos específicos Ca2 dinâmica em plântulas de Arabidopsis, e por imagem confocal de citosólica e Ca 2 + dinâmica nuclear em células das raízes de Arabidopsis que expressam Caso 12 luminescência de imagem de FAS pode ser adaptado para analisar induzidas pelo stress Ca2 dinâmica em plantas ou tecidos intactos não descritos aqui, ou para pesquisar populações de plantas de Arabidopsis mutantes por mutagénese com o stress alterado induzido Ca2 sinais. A configuração da imagem de células vivas de Ca2 + pode ser adaptado para analisar Ca2 + dinâmica em diferentes compartimentos subcelluar ou em diferentes tipos de células utilizando outros indicadores de Ca2 +.

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			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
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			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
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			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
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			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
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			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
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		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
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			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
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			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
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			<td height="21" style="height:21px;width:356px;">sucrose</td>
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			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
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			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
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measuring spatial and temporal ca2+ signals in arabidopsis plants

Sinais ambientais e de desenvolvimento induzir oscilações de Ca2 + em células de plantas. Espaço-temporais de Ca2 + padrões específicos de estímulo são detectados por proteínas Ca2 + ligação celulares que iniciam Ca2 + cascatas de sinalização. No entanto, ainda sabemos pouco sobre como estímulo sinais de Ca 2 + específico são gerados. A especificidade de um sinal de Ca2 + pode ser atribuído à regulação sofisticados das actividades dos canais de Ca2 + e / ou transportadores, em resposta a um dado estímulo. Para identificar estes componentes celulares e compreender as suas funções, é essencial a utilização de sistemas que permitem a gravação sensível e robusto de sinais de Ca 2 + em ambos os tecidos e níveis celulares. Codificados geneticamente Ca2 indicadores que são direcionados para diferentes compartimentos celulares têm proporcionado uma plataforma para células vivas imagem confocal de celulares Ca2 + sinais. Aqui descrevemos instruçãos para a utilização de dois sistemas de detecção de Ca 2 +: aequorina baseado FAS (mudas adesivas película luminescência) de Ca2 + e de imagiologia case12 confocal de fluorescência de células vivas com base de Ca2 + de imagem. Imaging luminescência usando o sistema FAS fornece uma detecção simples, robusto e sensível de Ca2 + sinais espaciais e temporais em nível tecidual, enquanto células vivas imagem confocal usando case12 fornece detecção simultânea de citosólica e Ca2 + sinais nucleares em uma resolução alta.

Manitol, NaCl e H 2 O 2 foram usados ​​como proxies para a desidratação, de sal e de estresse oxidativo estímulos, respectivamente. Para verificar se o ião de metal pesado Cu2 + sinergiza com qualquer destes três estímulos de stress, comparou-se a resposta de Ca 2 + a cada estímulo na presença ou ausência de Cu2 +. Como mostrado na Figura 2, o FAS luminescência imagiologia revelou que as plântulas Arabidoposis responderam diferentemen...

Watch this Scientific Journal Video about Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants at JoVE.com

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Sinalização de Ca2 + regula diversos processos biológicos em plantas. Aqui apresentamos abordagens para monitorar o estresse abiótico induzida Ca espacial e temporal 2 + sinais em células e tecidos, utilizando os codificados geneticamente Ca2 indicadores aequorin ou case12 Arabidopsis.

Medindo espacial e temporal Ca<sup&gt; 2 +</sup&gt; Sinais em Arabidopsis Plantas

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

Research

JoVE Journal

Biology

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

12.0K visualizações.  Purdue University.  Sinalização de Ca2 + regula diversos processos biológicos em plantas. Aqui apresentamos abordagens para monitorar o estresse abiótico induzida Ca espacial e temporal 2 + sinais em células e tecidos, utilizando os codificados geneticamente Ca2 indicadores aequorin ou case12 Arabidopsis. 

Vídeo: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

Modelagem de Membranas Biológicas, com placas de circuito e medição de sinais elétricos em Axônios: Exercícios Laboratório Student

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Biologia

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice Cap: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Medindo intracelular de Ca<sup&gt; 2 +</sup&gt; Mudanças no esperma humano, utilizando quatro técnicas: fluorometria convencional, Parado Fluorometria fluxo, citometria de fluxo e única célula de imagem

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automated Análise de Ca dinâmico<sup&gt; 2 +</sup&gt; Sinais em seqüências de imagens

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Gravação em tempo real na Vivo de Arabidopsis sinais de cálcio durante a alimentação de insetos usando um biossensor fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Aplicações de mapeamento de espaço-temporal e técnicas de análise de partículas para quantificar intracelular Ca2 + sinalização em Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Microdissection laser RNA-Sequenciamento para Análise de Expressão Genética Espacial e Temporal-Específica do Tecido em Plantas

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Imagens em campo amplo e em tempo real de sinais de feridas locais e sistêmicas em Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Um método de inoculação fácil e flexível para avaliar com precisão os fenótipos de infecção por oídio de Arabidopsis e outras plantas

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...