Os autores gostariam de agradecer as Dras. Heping Cheng, Huiliang Zhang e Stephen Kolwicz por seus comentários e apoio técnico no desenvolvimento deste método. Este estudo foi suportado por concessões do NIH e da Scientist Desenvolvimento Grant da American Heart Association para WW.

1. Experiência Preparação <ol><li> Preparar a solução isotónica salina equilibrada (50 ml) contendo: 140 mM de NaCl, 5 mM de KCl, 2,5 mM CaCl 2, 2 mM MgCl 2 e 10 mM de HEPES (pH 7,2) para a visualização in vivo do músculo esquelético. </li><li> Prepare 1 litro de tampão de Krebs-Henseleit (KHB) contendo: NaCl 118 mM, KCl 5,3 mM, MgSO4 1,2 mM, EDTA 0,5 mM e 25 mM de NaHCO3. </li><li> Bolha KHB com gás contendo 95% de O2 / 5% de CO 2 durante 10-15 minutos antes da adição de 2 mM de CaCl2. Adicionar substratos metabólicos (por exemplo, 10 mM de glucose e 0,5 mM de piruvato). </li><li> Prepare os instrumentos cirúrgicos com a esterilização a braçadeira, tesouras e fórceps em etanol 70% e, em seguida, enxaguar em esterilizado DDH 2 O. </li><li> Ligue o sistema de perfusão do coração, ajustar a temperatura do banho de água e circulador, defina a velocidade da bomba peristáltica a 6 ml / min. </li><li> Bolha KHB com 95% de O2/ 5% de CO 2 de gás, e controlar a taxa de fluxo e temperatura (37 ° C, por uma sonda de temperatura de fibra óptica) do efluente. O sistema precisa de cerca de 20 min para o gás ea temperatura de equilíbrio. </li></ol> 2. Imagem confocal dos músculos esqueléticos Em Vivo <ol><li> Anestesiar rato com pentobarbital (80 mg / kg, ip). O animal vai chegar a anestesia cirúrgica (sem resposta aos pés pitada) dentro de 10-15 min e permanecer neste estado por 1-1,5 h, suficiente para o vivo de imagens em dos músculos esqueléticos. </li><li> Retire os pêlos em um dos membros posteriores e esterilizar a pele com etanol a 70%. </li><li> Realizar uma incisão na pele ao longo do lado exterior do membro de expor os músculos gastrocnemius. </li><li> Pegue o epimísio delicadamente com uma pinça afiada e fazer uma incisão através dela com uma tesoura. Além disso dissecar para remover o epimísio e expor as fibras musculares por baixo. </li><li> Para in vivo carregamento de TMRM no esqueléticomuscular, incluem TMRM (500 nM) em solução de sal equilibrada isotónica imergir músculo durante 30 minutos e, em seguida lavar com uma solução isenta de indicador. </li><li> Colocar o rato sobre o seu lado no microscópio confocal (Zeiss LSM 510) fase e restringir o membro posterior em uma posição em que o músculo esquelético é exposta virada contra a lamela que forma o fundo da câmara. A lamela é entre o tecido muscular e o objectivo invertido (óleo de 40X). </li><li> Pressione a perna para baixo com cuidado para fazer contato apertado entre o tecido ea lamela. Mergulhe os músculos expostos em solução salina balanceada isotônica. </li><li> Grave duas dimensões (2D, xy) imagens confocal, a uma taxa de amostragem de um segundo por quadro. A intensidade de cada pixel é digitalizado em profundidade de 8 bits. Normalmente, uma varredura de série contém 100 quadros. </li><li> Coletar imagens seqüenciais por primeiro emocionante a 405 nm e coleta de&gt; 505 nm a 488 nm, em seguida, durante a coleta de&gt; 505 nm de comprimento de onda para a dupla excitation imagens de mt-cpYFP. Use excitação seqüencial a 405, 488 e 543 nm, e recolher emissão em 505-545, 505-545 e&gt; 560 nm, respectivamente, para o triplo do comprimento de onda de excitação de imagem de mt-cpYFP e TMRM. </li></ol> 3. Imagem confocal de perfusão Rato Coração <ol><li> Imediatamente após o vivo de imagens em dos músculos esqueléticos, injetar o mouse com 200 unidades de heparina (ip). Dez minutos depois, a eutanásia o mouse por toracotomia e rapidamente remover o coração com os pulmões eo timo ligadas a ele. </li><li> Remover rapidamente o pulmão em tampão gelado. Identificar os lóbulos do timo e suavemente descascar para trás para expor a aorta ascendente. </li><li> Remover o timo e isolar da aorta com cuidado de remover todo o tecido circundante. </li><li> Faça um corte na extremidade superior da aorta ascendente antes do primeiro ramo do arco aórtico. </li><li> Segurar a parede da aorta com cuidado com duas pinças de micro sutura para expor o lúmen e colocar cuidadosamente a aorta para o cannula (PE50 tubo). A aorta é mantida no lugar com uma pinça micro navio enquanto as suturas são rapidamente amarrado ao redor da aorta. </li><li> Retire o grampo, verifique cuidadosamente a cânula com uma pinça para garantir que a ponta da cânula está acima da raiz da aorta. Laços adicionais são adicionados como necessário para manter o coração no lugar. </li><li> Ligar a bomba peristáltica e perfundir o coração a 1 ml / min. O coração vai continuar batendo em perfusão. </li><li> Ajuste a posição do sistema de perfusão e colocar o coração no palco microscópio. A fase tem um adaptador que permite o aquecimento da câmara que contém o coração. </li><li> Adicionar 1 ml de solução de perfusão KHB na câmara para submergir parcialmente o coração. Monitorizar a temperatura da câmara a 37 ° C. Retirar efluente da câmara usando uma bomba peristáltica. </li><li> Aumentar a velocidade de bomba peristáltica gradualmente para fornecer fluxo suficiente (aproximadamente 2 ml / min) para o coração. </li><li> Após 10 minutos de estabilização, perfundir o heart com 10 uM e blebbistatin 100-500 nM TMRM. Batimento cardíaco vai abrandar depois de 10 min. </li><li> Aplicar uma leve pressão sobre o coração para assegurar um contacto apertado do coração com a lamela na parte inferior da câmara, e para suprimir ainda mais batimentos cardíacos. </li><li> Siga o mesmo procedimento para a imagem confocal de miocárdio íntegro, como descrito no passo 2.8 acima. Ajustar cuidadosamente o plano focal para revelar a imagem mais nítida possível. </li></ol> 4. Processamento de Imagem e Análise de Dados <ol><li> Use as ferramentas fornecidas pelo módulo &quot;Análise fisiológica&quot; do software confocal para analisar único mitocondriais flashes e membrana possíveis mudanças. Este módulo é freqüentemente incluída na imagem adquirir software do sistema confocal e permite a determinação de certas áreas da imagem, bem como a saída de intensidade de fluorescência com rótulos de tempo. </li><li> Abra o banco de dados e, em seguida, o arquivo de imagem 2D de série a ser analisado. </li><li> Clique no botão &quot;Regiãode interesse (ROI) significa &quot;para mudar para a&quot; média de ROIs &quot;mode. Clique no botão&quot; região de interesse (ROI) significa &quot;para mudar para o&quot; modo média de ROI &quot;. </li><li> Desative a exibição de outros canais, exceto o canal de cpYFP 488 nm para a seleção dos flashes. </li><li> Aumentar a imagem e manualmente mover barra deslizante para reproduzir as imagens em 2D série. </li><li> Identificar único superóxido mitocondrial flashes por localizar o local onde sinais fluorescentes aumenta transitoriamente. Use a ferramenta ROI apropriado para marcar os flashes. O traço mostrando dependente do tempo a mudança de fluorescência de cada ROI vai aparecer ao lado da imagem. </li><li> Depois de selecionar todos os flashes, ligue a exibição de outros canais. Selecione um ROI na imagem exterior da célula para o sinal de subtração de fundo. Saída da fluorescência média de cada ROI juntamente com as etiquetas de tempo. </li><li> Registre o número de flashes em cada um dos arquivos de imagem de varredura de série juntamente com o tempo ea área de varreduraa célula. Use o Excel para calcular a frequência de flash como número de flashes por 100 seg / 1000 um 2 Área do celular. </li><li> Use um programa desenvolvido sob medida escrito em Interactive Data Language (IDL, ResearchSystems) para calcular os parâmetros de cada Flash (amplitude, tempo de pico e tempo de decaimento). Software IDL está disponível comercialmente eo programa desenvolvido sob medida para a análise de parâmetros de flash pode ser obtida a partir do autor, mediante solicitação. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Imagiologia eventos mitocondriais individuais em animal vivo ou órgãos perfundidos tem vantagem significativa em relação aos métodos tradicionais de avaliação da função mitocondrial 17,19,21,22,24,25. A técnica aqui descrita pode realizar em tempo real a determinação in situ da função mitocondrial em condições fisiológicas reais na resolução subcelular. Isto é particularmente útil, quando combinada com outras medidas, para estudar sistematicamente o papel da mitocôndria...

As mitocôndrias têm um papel central na bioenergética celular, sinalização de radicais livres, homeostase redox, regulação iônica e determinação do destino da célula 1,2. As mitocôndrias disfunção muitas vezes acompanha e sustenta a patogênese de doenças 3-6. Especialmente nos sistemas musculares, tais como o coração e músculos esqueléticos, respiração mitocondrial fornece a maior parte do ATP para apoiar a regulação atempada de cálcio intracelular e robusto 7,8 desenvolvimento de força. Estes músculos possuem um grande número de mitocôndrias, que muitas vezes ocupam até 20-40% do volume total de células e são &quot;fixos&quot; entre miofilamentos 2. Apesar de numerosos estudos, a nossa compreensão da regulação da função mitocondrial, especificamente in vivo e em condições fisiologicamente relevantes, é limitado. Uma das razões é que a maioria dos métodos desenvolvidos para avaliar a função mitocondrial confiar em VITRo ou ex vivo abordagens, tais como monitoramento do consumo de oxigênio de mitocôndrias isoladas suplementadas com substratos artificiais, ea determinação indireta da função mitocondrial através da morfologia (microscopia de elétrons, por exemplo), a atividade da enzima (por exemplo, atividade aconitase), ou os níveis de ATP intracelular 9-11 . Recentemente, pequenos indicadores fluorescentes molécula com enriquecimento relativo mitocondrial têm sido aplicados para fornecer um vislumbre dos sinais mitocondriais, incluindo o potencial de membrana, cálcio e espécies reativas de oxigênio (ROS), em células intactas 11-13. Além disso, vários de proteína fluorescente verde (GFP) e indicadores redox baseado ROS foram desenvolvidos para obter uma avaliação mais específica do redox intracelular compartimentada ou ROS sinaliza 14-16. Entre isto, foi desenvolvido um indicador geneticamente codificado superóxido, a proteína fluorescente amarela permutada circular, e targeted-lo para dentro da mitocôndria (mt-cpYFP) 17. mt-cpYFP pode ser excitado a 405 ou 488 nm, com dois picos de emissão de 515 nm. A emissão a 488 nm de excitação é especificamente responsivo a superóxido como mostrado pela anterior in vitro e in vivo calibrações 17,18. A emissão a 405 nm de excitação é utilizado como controle interno (consulte a Figura 1 da Ref. 17 para obter informações detalhadas sobre os espectros de emissão e excitação de mt-cpYFP sob várias condições). Com time-lapse confocal de imagem, este indicador detecta estourando eventos produção de superóxido dismutase, nomeado flashes, em mitocôndrias isolados de células intactas. Flash Superoxide serve como uma função composta de respiração mitocondrial, a despolarização da membrana mitocondrial transitória acompanhamento e produção de ROS 17-20. Recentemente, gerou os pan-tecido mt-cpYFP camundongos transgênicos usando o vector pUC-CAGGS-mt-cpYFP 17,19 em C57/BL6 fundo e verificar as expressões fortesção deste indicador no coração, músculos esqueléticos e outros tecidos (Figura 2). Os camundongos transgênicos estarão disponíveis para os investigadores acadêmicos interessados ​​mediante solicitação e aprovação MTA pela Universidade de Washington. Neste estudo, descrevemos em imagem in situ de superóxido flashes em Langendorff coração perfundido, bem como in vivo de imagens de eventos de flash em músculos esqueléticos de camundongos transgênicos mt-cpYFP anestesiados 17,19. Esta tecnologia permite o monitoramento em tempo real de eventos de produção ROS único mitocondrial em uma condição fisiologicamente relevante ou in vivo 21,22. Também é possível usar o sistema para controlar outros parâmetros individuais mitocondriais, tais como o potencial de membrana e de cálcio com indicadores fluorescentes adequadas. Avaliação Além disso, simultânea ou paralela da função mitocondrial com eventos intracelulares (por exemplo, transientes de cálcio) ou de função cardíaca (por exemplo,. Fração de ejeção) pode ser alcançado. Perturbações patológicas, tais como a isquemia e reperfusão, pode ser aplicada ao coração perfundido para avaliar o impacto do stress sobre a função mitocondrial único no miocárdio intacta.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Toronto Research Chemicals</td>
			<td>B592500</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Acros Organics</td>
			<td>AC34961-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H7006-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#8226;6H2O</td>
			<td>Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M1880-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S8282-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6014-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256-25</td>
			<td></td>
		</tr>
		<tr>
			<td>TMRM</td>
			<td>Invitrogen</td>
			<td>T-668</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Confocal Line Scanning Microscope (LSM 510 Meta, Zeiss), software version 4.2 SP1 including &#34;Physiological Analysis&#34; module.</td>
		</tr>
	</tbody>
</table>

confocal imaging of single mitochondrial superoxide flashes in intact heart or in vivo

Mitocôndria é uma organela intracelular crítico responsável pela produção de energia e de sinalização intracelular em sistemas eucariotas. Disfunção mitocondrial, muitas vezes acompanha e contribui para a doença humana. Maioria das abordagens que têm sido desenvolvidas para avaliar a função mitocondrial e disfunção são baseados na produção in vitro ou ex vivo, as medições. Os resultados desses experimentos têm capacidade para determinar a função mitocondrial in vivo limitado. Aqui, descrevemos uma nova abordagem que utiliza microscopia confocal para a imagem de tecidos intactos em aminals ao vivo, o que permite a avaliação da função mitocondrial único de uma forma em tempo real in vivo. Em primeiro lugar, gerar camundongos transgênicos expressando o indicador de superóxido mitocondrial alvo, a proteína fluorescente amarela permuta circular (mt-cpYFP). Mt-cpYFP anestesiados mouse é fixo em um adaptador de estágio feito por encomenda e as imagens de lapso de tempo são tomadas from os músculos esqueléticos expostas do membro posterior. O mouse é posteriormente sacrificado eo coração está configurado para Langendorff perfusão com soluções fisiológicas a 37 ° C. O coração perfundido está posicionado em uma câmara especial no palco microscópio confocal e uma leve pressão é aplicada para imobilizar o coração e suprimir batimento cardíaco artefato movimento induzido. Superóxido flashes são detectados pelo tempo real de imagens 2D confocal com uma frequência de um quadro por segundo. A solução de perfusão podem ser modificadas para conter diferentes substratos de respiração ou outros indicadores fluorescentes. A perfusão pode também ser ajustado para produzir modelos de doenças tais como a isquemia e reperfusão. Esta técnica é uma abordagem única para a determinação da função de simples mitocôndria em tecidos intactos e in vivo.

De acordo com este protocolo, imagem in vivo de eventos mitocondriais único pode ser feito em músculos esqueléticos de ratos anestesiados, seguido por em imagem situ no coração perfusão (Figura 1). A configuração ideal das condições de imagem garante imagens nítidas dos tecidos musculares intactas e com resolução mitocôndria única (Figura 2). TMRM é muitas vezes usado para verificar a localização de mt-cpYFP e deve apresentar um padrão de so...

Watch this Scientific Journal Video about Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo at JoVE.com

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Microscopia confocal é aplicada para imagens individuais em eventos mitocondriais coração perfundido ou músculos esqueléticos em animal vivo. Monitoramento em tempo real de processos mitocondriais único, como superóxido flashes e membrana possíveis flutuações permite a avaliação da função mitocondrial em um contexto fisiologicamente relevante e durante perturbações patológicas.

Imagem confocal de Solteiro mitocondriais superóxido Pisca em coração intacto ou<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

confocal-imaging-single-mitochondrial-superoxide-flashes-intact-heart

Research

JoVE Journal

Biology

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

14.5K visualizações.  University of Washington.  Microscopia confocal é aplicada para imagens individuais em eventos mitocondriais coração perfundido ou músculos esqueléticos em animal vivo. Monitoramento em tempo real de processos mitocondriais único, como superóxido flashes e membrana possíveis flutuações permite a avaliação da função mitocondrial em um contexto fisiologicamente relevante e durante perturbações patológicas. 

Vídeo: Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Eletroporação única célula in vivo dentro do cérebro intacto desenvolvimento

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Biologia

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Gravações de uma única célula de sucção de cones fotorreceptores rato

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluorescência baseada em Medição da loja operada entrada de cálcio em células vivas: a partir de células cancerosas cultivadas em fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Molécula única imagem da regulação gênica<em&gt; Em vivo</em&gt; Uso de ativação Cotranslational por clivagem (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

De alta resolução Respirometria para avaliar a função mitocondrial em permeabilizadas e células intactas

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparação e imagem longitudinal do coração Função In Vivo Usando Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.