Este trabalho foi financiado em parte pelo Instituto Nacional de Bolsas de Saúde HL-085887, HL-092992, S10RR027535 e MOP-93676.

1. Dissection Vessel and Imaging <ol><li> Tecido Colheita de suínos juvenis domésticos, conforme descrito no Martens et al 27. Coloque ventrículos direito suínos colhidas em um polidimetilsiloxano (PDMS) prato dissecção inferior contendo tampão HEPES solução salina fisiológica (PSS). <ol><li> Com o auxílio de um microscópio estereoscópico, dissecar e remover um segmento da artéria coronária descendente anterior da artéria coronária (~ 8 milímetros de comprimento, 0,5 mm de diâmetro) de tecido circundante, utilizando um fórceps e tesouras mola através da remoção do segmento de vaso a partir das camadas de tecido cardíaco circundantes. NOTA: Tome cuidado para não perfurar a parede do vaso. </li><li> Coloque um bloco PDMS (1 x 0,5 x 0,5 cm) para o prato de dissecação, e usar uma agulha para fixá-lo para o fundo. Cortar um diâmetro de fio de tungstênio, aproximadamente, 40 mM em doze segmentos de comprimento ~ 0,3 centímetros, formando micropins, e colocá-las no prato. Usando fórceps, fixar uma extremidade do segmento do vaso para o bloco com uma micropin. </li><li> Insira com cuidado tesoura pequena mola no lúmen do vaso e cortar longitudinalmente de um lado do navio para abri-lo completamente. Oriente o segmento navio abriu com o endotélio para cima. </li><li> Use as micropins restantes para proteger as fronteiras do segmento de vaso aberto para bloquear tais que a embarcação forma um retângulo plano. NOTA: topos Micropin devem ser dobrados a 90 º e inserido até nivelada com a superfície do bloco. Cuidados devem ser tomados para esticar a preparação navio sem hostes; largura final deve ser ~ 1,5 x a largura não esticada partida. </li><li> Prepara-se uma pequena quantidade (~ 1 ml) da solução de Fluo-4 AM carregamento por mistura de Fluo-4 AM (10 fiM) dissolvido em DMSO com plurico (0,03%) em um tampão HEPES PSS (contendo em mM: NaCl 134, KCl 6 , 1 MgCl, 10 HEPES, 10 glucose), e inserir o bloco inteiro na solução de carga para, aproximadamente, 40 minutos à temperatura ambiente no escuro. </li><li> Após carga, lava-se a blocos de tampão HEPESPSS para 5-10 min. </li><li> Monte o bloco para 50-100 ^ M espaçadores de espessura em uma câmara inferior lamela contendo tampão HEPES PSS. NOTA: pinos de metal podem ser utilizados como separadores e assegurar o segmento navio está voltada para baixo e não tocar os espaçadores. </li><li> Coloque a câmara no palco de um microscópio invertido equipado para imagem confocal, e concentrar-se sobre a camada de células endoteliais. </li><li> Sequências de imagens de lapso de tempo de captura de fluorescência relativa basal para ~ 3 min com ampliação de 20x e uma taxa de quadros de ~ 8 frames por segundo utilizando imagem confocal software seqüência de aquisição. </li><li> Após cerca de 3 minutos de gravação de fluorescência basal, substituir solução tamponada HEPES PSS com o mesmo volume (~ 1 ml) da substância P (100 pM) dissolvido em tampão HEPES PSS, e uma ficha de autopropulsão 3 min. </li></ol></li></ol> 2. Análise automatizada <ol><li> Renderização seqüência (s) imagem da atividade fluorescente de cálcio a partir de software de aquisição confocal como de 8 bits, grayscale &#39;. tif&#39; arquivos de formato sem informação de escala. </li><li> Abrir ImageJ, clique em &#39;open&#39; no menu arquivo e selecione a imagem seqüência apropriada na janela do Explorer para exibir a seqüência (s) imagem em ImageJ. </li><li> Determinar um diâmetro ROI apropriado usando a ferramenta ROI rectangular para estimar os limites superiores e inferiores da distribuição espacial da actividade dentro da sequência (s) da imagem. NOTA: O diâmetro médio retangular é uma escolha adequada para diâmetro ROI. </li><li> Crie uma nova pasta no disco rígido do computador, e adicione a seqüência (s) imagem para o diretório da pasta. </li><li> No ImageJ, clique em janela &#39;plugins&#39; e clique em &#39;LC_Pro&#39; para iniciar a análise. </li><li> Digite o valor do diâmetro ROI e selecione o valor limiar de filtro (ou 0,05 ou 0,01). </li><li> Clique na caixa de seleção &quot;tratamento de drogas&quot; e insira os valores de ponto de tempo imediatamente antes e depois que a droga foi adicionado (em segundos). </li><li> Clique em &#39;ok&#39;, edigite o diretório sequência de imagens para a janela do explorador de arquivos. </li></ol> 3. Saída gráfica <ol><li> Baixe a versão 3.0.2 a partir de R <a href="http://www.r-project.org/">http://www.r-project.org/</a> . </li><li> Abra a versão de R. 32 bits </li><li> Clique em &quot;Arquivo&quot; e depois &quot;script aberto &#39;e selecione o script de R&#39; traceplot.R &#39;para gerar relatórios experimentais gráficas. </li><li> Instale a calibrar e gplots pacotes, selecionando &#39;pacotes&#39;, &#39;instalar pacotes &quot;, e selecionando os pacotes justificativo. </li><li> Clique em &#39;arquivo&#39;, depois &#39;altere o diretório&#39; comando e usar a janela do explorer para selecionar o diretório que corresponde à saída de análise LC_Pro. </li><li> Clique na janela de script e, em seguida, o &quot;executar todos&quot; comando para executar o script. </li></ol>

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Decodificando complexos Ca 2 + sinais a nível celular e multicelular exigirá abordagens experimentais e analíticos rigorosos. Aqui, é descrito um método em que a sequência de tempo de imagem confocal resolvidos de Ca 2 + dependente de fluorescência são submetidas a uma análise automatizado que identifica e quantifica a Ca 2 + sinais estatisticamente relevantes dentro de campos celulares intactos no caso concreto apresentado, um segmento da artéria foi isolado de coração de po...

Ca 2 + é um segundo mensageiro ubíquo molécula sinalizadora e citosólicas de Ca2 + níveis são altamente regulamentados. Ca 2 + intracelular sinais são complexas e incluem transientes isoladas, oscilações, e as ondas de propagação 1-4. Controle espacial e temporal da Ca 2 + é pensado para ser a base especificidade sinal fisiológico e, portanto, a análise de Ca 2 + padrões de sinal é de considerável interesse para os investigadores em vários campos 5. Corantes de Ca 2 +, como indicador de Fluo-4 e de Fura-2 são normalmente utilizados para medir intracelulares de Ca 2 + sinais com microscopia de fluorescência 5-12. Normalmente, os temporais Ca 2 + sinais são avaliados como alterações dependentes do tempo em média de fluorescência dentro de uma área definida pelo usuário, ou região de interesse (ROI) 5,6,13 - 16. Atualmente, a análise de ROI manual é tanto demorado e trabalho emintensiva, porque requer que os usuários identifiquem muitos ROIs e realizar cálculos repetitivos 17 - 19. Essas técnicas também podem ser sujeitos a considerável erro do usuário, incluindo a introdução de modos de sinal artificiais e exclusão de baixa amplitude ou sinais difusos 18,20. Algoritmos automatizados de detecção de ROI previamente implementada utilizando uma variedade de métodos estatísticos para determinar a colocação óptima de ROI, mas eles têm sido geralmente limitados à análise de varrimento de linha ou pseudo linha de imagens digitalizadas, que limita a análise a um único dimensão espacial no tempo 17, 19-22. Além disso, muitos algoritmos existentes não são suficientes para abranger a diversidade de Ca 2 + eventos de liberação, que vão desde, transientes localizadas periódicas às ondas que se propagam 23,24. Avaliação abrangente dos fisiológicos de Ca 2 + sinais muitas vezes é ainda mais complicada pela presença de Artif imagem significativaagir que confunde sinal de discriminação de ruído em muitos sistemas experimentais. Anteriormente, um algoritmo de detecção de solução automatizada ROI para Ca 2 + sinal de detecção transitória, implementado como um plugin para o software ImageJ NIH (National Institutes of Health, Bethesda, MD), foi desenvolvido e validado 25,26. Este algoritmo, chamado LC_Pro, foi projetado para identificar e analisar ROIs abrangendo Ca 2 + transientes de sinal em sequências de imagens de lapso de tempo bidimensionais. Aqui um protocolo experimental e demonstração prática representante de uma aplicação do algoritmo no endotélio das artérias coronárias de suínos é fornecido, com pós-processamento adicional, usando o open source software de processamento estatístico R para gerar a saída gráfica utilizável.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissection dish</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#08-772-70</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polydimethylsiloxane (PDMS)</td>
			<td style="width:71px;">Fisher Sci</td>
			<td style="width:119px;">#NC9644388</td>
			<td>elastomer kit, must be molded into dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES-buffered PSS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">#H3375-250G</td>
			<td>HEPES acid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">stereomicroscope</td>
			<td style="width:71px;">Nikon Inst.</td>
			<td style="width:119px;">#MNA42000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">forceps</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#11223-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">spring scissors</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">tungsten wire</td>
			<td style="width:71px;">Scientific Inst Svcs</td>
			<td style="width:119px;">#406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4 AM</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#F-14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pluronic F-127</td>
			<td style="width:71px;">Life Tech.</td>
			<td style="width:119px;">#P3000MP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">metal pins</td>
			<td style="width:71px;">Fine Science Tools</td>
			<td style="width:119px;">#26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cover-glass bottom chamber</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Custom designed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spinning disc confocal microscope</td>
			<td style="width:71px;">Perkin Elmer</td>
			<td style="width:119px;">RS-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imageJ software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/download.html">download at: http://rsbweb.nih.gov/ij/download.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC_Pro plugin for imageJ</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html">download at: http://rsbweb.nih.gov/ij/plugins/lc-pro/index.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R software</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="http://www.r-project.org/">download at: http://www.r-project.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">R traceplot script</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td><a href="https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing">download at: https://docs.google.com/file/d/0B-PSp1D9e2fjV3NIcGppUzkxdEk/edit?usp=sharing</a></td>
		</tr>
	</tbody>
</table>

automated analysis of dynamic ca2+ signals in image sequences

Ca 2 + intracelular sinais são vulgarmente estudada com corantes de Ca 2 + indicadoras fluorescentes e as técnicas de microscopia. No entanto, a análise quantitativa de Ca 2 + dados de imagem é demorado e sujeito a viés. Algoritmos de análise de sinal automatizado baseado na região de interesse (ROI) de detecção têm sido implementadas para medições de varredura de linha unidimensional, mas não há nenhum algoritmo atual que integra a identificação otimizado e análise de ROI em sequências de imagens bidimensionais. Aqui um algoritmo para rápida aquisição e análise de ROI em sequências de imagens é descrito. Ele utiliza elipses caber aos sinais filtrados de ruído, a fim de determinar a colocação ROI ideal, e calcula Ca 2 + parâmetros do sinal de amplitude, duração e distribuição espacial. Este algoritmo foi implementado como um plug-in gratuito para software ImageJ (NIH). Juntamente com os scripts de análise escritos para o open source software de processamento estatístico R,Esta abordagem proporciona um gasoduto de alta capacidade para a realização de análise estatística rápida da produção experimental. Os autores sugerem que o uso deste protocolo de análise levará a uma caracterização mais completa e imparcial do fisiológica Ca 2 + de sinalização.

Um algoritmo de costume, LC_Pro, foi desenvolvido e implementado, a fim de realizar a análise automatizada de Ca 2 + dinâmicas em sequências de imagem confocal. Como representado na Figura 1, o algoritmo utiliza módulos de processamento sequenciais que A) detectar e locais de pista de dinâmica de Ca 2 + mudar acima estatística (p &lt;0,01) o ruído, B) definir as regiões de interesse (ROI) automaticamente em centros de sítio activo, e C) calcular intensidades médias de flu...

Watch this Scientific Journal Video about Automated Analysis of Dynamic Ca2+ Signals in Image Sequences at JoVE.com

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Aqui um romance região do protocolo de análise de juros com base na classificação elipses melhor ajuste atribuídos a regiões de sinal positivo dentro de sequências de imagens de lapso de tempo bidimensional é demonstrada. Este algoritmo pode permitir que os investigadores a analisar exaustivamente sinais fisiológicos Ca + 2 com entrada mínima do usuário e preconceito.

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+ ...

automated-analysis-of-dynamic-ca2-signals-in-image-sequences

Research

JoVE Journal

Biology

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

17.0K visualizações.  University of South Alabama.  Aqui um romance região do protocolo de análise de juros com base na classificação elipses melhor ajuste atribuídos a regiões de sinal positivo dentro de sequências de imagens de lapso de tempo bidimensional é demonstrada. Este algoritmo pode permitir que os investigadores a analisar exaustivamente sinais fisiológicos Ca + 2 com entrada mínima do usuário e preconceito. 

Vídeo: Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

Aplicação de Light-resina adesivo dental para Eletrodos de montagem ou Sondas microdiálise em Experimentos crônica

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Biologia

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semi-automático de análise de pulsação óptica de pequenos corações

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

Transgénese mosaico Zebrafish de Avaliação Sequências Enhancer

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica e Análise de Imagem Dinâmica de Estudos de Expressão Gênica em Tecidos Vegetais

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Técnicas de imagem Ca 2 + Sinalização no esperma humano

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / Flash Rotulagem e Análise de Imagem Sensor de Proteínas em Células Tetracysteine

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

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+ ...

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, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Protocolo de varredura do relógio para análise de imagem: plugins ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...