Apoio Grant: NIH CA165263, CA150197 NIH, NIH CA150197S1 (HJ Kung), NIH CA150197S1 (CA Changou), NSF PHY-0120999 Centro de Biofotônica da Ciência e Tecnologia (DL Wolfson, FYS Chuang), DOD PC073420 (RJ Negrito), a pesquisa Conselho da Noruega, Leiv Eiriksson viagem Grant 209286/F11 (BS Ahluwalia). HJ Kung também agradece o apoio do Fundo Endowment Cancer Comunidade Auburn. RJ Negrito também reconhece o apoio da J. McDonald investidura. Agradecemos ao Dr. Jenny Wei-Jen Kung e Dr. Bor-wen Wu em DesigneRx para generosa oferta de ADI.

1. Parte 1: Cultura de Células e Rotulagem Imuno-fluorescência <ol><li> Cresça CWR22 RV1 células tumorais da próstata humana em lamelas de vidro (# 1,5 ou 170 m de espessura), colocadas em placas de 6 poços, com meio RPMI (Mediatech, VA) contendo 10% de soro fetal bovino (FBS) e 1% de penicilina / estreptomicina / glutamina. </li><li> Induzir autofagia amostras seleccionadas por tratamento com arginina deiminase (ADI, 0,3 ug / ml) em solução salina tamponada com fosfato (PBS). </li><li> Fixar as células com paraformaldeído a 4% (PFA, Fisher Scientific, NH), diluídas em PBS, 100 ul por lamela, durante 10 min à TA. </li><li> Lave as células 3x com 1 ml de HBS / BSA. </li><li> Permeabilizar células com 0,05% de saponina (Sigma, MO) em tampão HEPES (HBS) / BSA, com 100 ul por lamela, durante 15 min à TA. </li><li> Antes de imuno-marcação, as amostras do bloco com 2,5% de caseína (Sigma, MO) em HBS / BSA, com 100 ul por lamela, durante 1 hora à temperatura ambiente. </li><li> Incubar as células fixadas com anticorpos primários diluídos em 2,5% de caseína / HBS / BSA, com 100 ul por coverslip, O / N a 4 ° C. </li><li> Lave as células 3x com 1 ml de HBS / BSA. </li><li> Incubar as células fixadas com anticorpos secundários diluídos em 2,5% de caseína / HBS / BSA, com 100 ul por lamela, durante 1 hora à temperatura ambiente. </li><li> Lave as células 3x com 1 ml de HBS / BSA. </li><li> Monte lamelas preparadas em lâminas de vidro para microscópio regulares com ouro SlowFade ou prolongar reagente antifade Gold (Invitrogen, CA), contendo 4 &#39;,6-diamidino-2-phenylindole (DAPI). </li><li> Bordas Seal lamela com unha polonês. Para obter os melhores resultados de imagem, dar tempo (de preferência O / N) para a mídia anti-fade a permear completamente as células. </li><li> Superfície lamela limpo com metanol e / ou clorofórmio. </li><li> Adicionar óleo de imersão (índice de refracção 1,520, quando imagiologia a 37 ° C, ou quando 1,516 imagiologia à TA) à 1,42 NA lente objectiva 60X (Olympus, Japão). </li><li> Posição corrediça na platina do microscópio e estabelecer o foco com as células de interesse. </li></ol> 2. Parte 2: Preparando Células para imagens ao vivo <ol><li> Crescer CWR22 RV1 células que expressam a proteína verde fluorescente acoplado Cadeia leve de 3 (LC3-GFP) em meio de cultura RPMI contendo 10% de FBS e 1% de antibióticos de 35 mm revestidas com placas de cultura de vidro com fundo de poli-d-lisina (MatTek, MA). As células devem ser colocadas em placas a uma densidade suficiente para facilitar a proliferação rápida, mas não tanto que as células são cobertas e aglutinado por o tempo de imagiologia. </li><li> Tratar amostras de células seleccionadas com ADI (0,3 ug / ml) em PBS. </li><li> Cerca de 1 hora antes da imagem, diluir 1,5 ul de Lyso Tracker Vermelho DND-99 (Invitrogen, CA), com 20 ml de RPMI. contendo 10% de FBS e 1% de antibióticos. Use soluções contendo ADI para amostras selecionadas. Aquecer todos os suportes a 37 ° C antes da adição à placa de cultura. </li><li> Incubar as células com meio RPMI contendo Lyso Tracker Vermelho DND-99 para 15-45 min a 37 ° C. </li><li> Cerca de 30 minutos antes da imagiologia, ligar o invólucro do ambiente EstaçãoMeteorológica e deixar equilibrar a 37 ° C e 5% de CO 2 (com humidifar IED). </li><li> Lavar as células com PBS e substitua com meio padrão RPMI contendo apenas 10% de FBS e 1% de antibióticos. Adicionar ADI para amostras conforme indicado. </li><li> Pratos de montagem 35 milímetros lamela com fundo de cultura em placa personalizada (Applied Precision, Inc., WA) e posição no palco microscópio. Use óleo de imersão (índice de refração 1,520) no 60X 1,42 NA lente objetiva, e posicionar o prato de cultura montado no palco. </li></ol> 3. Parte 3: Microscopia Deconvolution e Análise O protocolo nesta seção assume o uso do DeltaVision pessoal DV deconvolution microscópio e associados aplicação suíte Softworx (Applied Precision, Inc., WA). <ol><li> Ligue o sistema de microscópio, incluindo o controlador da estação de trabalho e de aquisição de instrumentos. Abra o aplicativo Resolve3D para inicializar o estágio do microscópio e ligue a fonte de luz xenon. Permitir 10 min para a fonte luminosa para aquecer e atingir condições estáveis. </li><li> Adicionegota de óleo de imersão (índice de refração 1,520 para amostras vivas a 37 ° C ou 1,516 para amostras fixas no RT) no 60X 1,42 NA objectiva, e um centro de espécime de slides (tampa de deslizamento para baixo) sobre a lente objetiva. </li><li> Usando qualquer iluminação de campo claro ou externa eo foco grosso botão de ajuste, levante lentamente a objetiva até que o talão de óleo de imersão faz contato com a tampa de vidro invertida. (A partir deste ponto, a camada de células deve entrar em foco em poucas voltas do foco fino botão de ajuste). Ao trabalhar com amostras fixadas sobre as lâminas, é recomendado para evitar a células dentro ou perto de quaisquer áreas com bolhas. Ao visualizar com amostras vivas no prato com fundo de vidro, evitar a movimentação da lente objetiva fora do limite da lamela de vidro. </li><li> Autophagosomes podem ser identificadas por um sinal de eGFP (verde). Os lisossomos são identificados por Lyso Tracker Red (amostra ao vivo) ou imunofluorescência anti-Lamp1 (amostra fixa). Núcleos de células são identificadas por DAPI raining (amostra fixa). </li><li> Selecione o campo de visão desejado. Idealmente, as células devem exibir boa sinal fluorescente em todos os canais apropriados, e suficientemente ligado e espalhar-se sobre a superfície da lamela de modo a que os conteúdos intracelulares é fácil de visualizar. Pequenos ajustes laterais em x, y, e z-focagem podem ser controladas por computador, utilizando a interface Acquire3D. Conjunto para binning 1x1 ou 2x2, dependendo campo de visão desejado (mais células pode ser vista no campo quando binning é definido como 2x2). </li><li> Para uma dada imagem através da barriga de uma célula, defina os parâmetros de exposição (por exemplo,% de transmissão de energia e tempo de exposição) de tal forma que a intensidade máxima de pixel não exceda 3.000 pontos. Geralmente, a% de transmissão deve ser ajustado tão baixo quanto possível, sem aumentar o tempo de exposição correspondente ao longo de 1 seg. Repita esse procedimento para cada cor de fluorescência para ser trabalhada e para cada campo separado de vista para obter as imagens da mais alta qualidade. </li><li> Defina o upper limites e inferiores do Z-pilha, ajustando o foco apenas ligeiramente ao longo do topo e da parte inferior da amostra de células, respectivamente. O número total de imagens de uma dada pilha Z será, assim, determinado por estes limites e por o espaçamento entre as camadas (valor padrão: 0,2 um). </li><li> Para minimizar artefatos de movimento durante a aquisição de imagem, recomendamos definir o modo de aquisição de imagem para &quot;comprimento de onda, então z-stack&quot; para amostras fixas, e &quot;z-stack depois de comprimento de onda&quot; para amostras vivas. </li><li> As imagens de fluorescência são deconvolved usando Softworx (Precision Aplicada, WA) e depois analisados ​​utilizando Volocity (Improvision, agora Perkin Elmer, MA). </li></ol> 4. Parte 4: Super-resolução, Structured-iluminação (OMX) Microscopia O protocolo nesta seção aplica-se ao uso da OMX Microscópio iluminação estruturada (Applied Precision, WA). <ol><li> Ligue a alimentação principal e lasers desejados (410 nm, 488 nm e / ou 532 nm). Aguarde 20 min para os lasers de ser estabilizado termicamente. </li><li> Adicione o óleo de imersão (índice de refração 1,516 para amostras fixas no RT) no 60X 1,42 NA lente objetiva. Se as bolhas são vistos na gota de óleo, limpe o objetivo e adicione o óleo novo. </li><li> Coloque amostra de slides no palco e, se necessário, permitir que 10 min para que as células se contentar em lamela. </li><li> Inicializar o programa de aquisição. Use iluminação de fluorescência para ajustar o foco até um contorno nítido da célula pode ser visto. (Deve ter cuidado em todos os momentos para minimizar a exposição das células a excitação de fluorescência, até a aquisição de imagem real). </li><li> Uma varredura de mosaico em espiral podem ser adquiridos para visualizar áreas maiores da amostra para selecionar células ou regiões de interesse. Recomenda-se usar tempos de exposição de curta duração (1-10 ms) e baixo consumo de energia de excitação (0,1-1% de transmissão) durante a digitalização da amostra ou encontrar alvos. </li><li> Identificar autofagossomas por fluorescência de GFP-LC3. Identificar lisossomos por Alexa Fluor 555 anti-LAMP(Proteína de membrana associada ao lisossoma) e núcleos de células utilizando DAPI (amostras fixas). </li><li> Escolha condições experimentais, incluindo comprimento de onda de excitação, a área de imagem (por exemplo, 512x512), espessura de pilha, tempo de exposição e taxa de transmissão laser. Para imagens de super-resolvida, verifique se a opção de iluminação estruturada está habilitado. Para a microscopia deconvolution na OMX, selecione a opção de iluminação convencional. </li><li> Para obter os melhores resultados de reconstrução, seleccionar as condições experimentais tais que a contagem de intensidade máxima está entre 10.000 e 15.000, durante a aquisição. Se fotodegradação é uma preocupação, a imagem latente numa menor exposição (contagens entre 5.000 e 10.000), podem ser usados. Idealmente, as contagens não deve diminuir mais do que um factor de dois durante toda a aquisição, e diminui em mais de um factor de quatro deve ser evitada. Se necessário, reduzir a espessura da pilha para assegurar uma contagem intensidade sustentada. A câmera satura a 64.000 contagens, então no máximointensidade nunca deve exceder esse valor. Para bons exemplos, encontramos as condições experimentais em 10 ~ 50 ms de tempo de exposição com transmissão do laser de 1%. São preferidos amostras brilhantes e fotoestável que pode ser visualizada, repetidamente. A mancha estável é necessária para time-lapse, imagens de super resolvidos. </li><li> Para reduzir o ruído, a velocidade de leitura de 95MHz (opção de velocidade média) e tempo de aquisição de 2 ms ou mais é recomendado. Aumento dos artefactos são obtidos na fotografada reconstituídas, quando a amostra se move durante a aquisição. Devido a este efeito, as exposições curtas são recomendados para as células não aderentes ou de movimento rápido. </li><li> Multicanal imagiologia pode ser realizado simultaneamente ou sequencialmente. Rendimentos de modo seqüencial menos cross-talk e é, portanto, recomendado. </li><li> Para reduzir a fotodegradação, é geralmente recomendado para a imagem com comprimentos de onda maiores primeiro excitação (532 nm, 488 nm, 410 nm e em seguida). No entanto, os resultados experimentais mostrados na Figura 3, essa ordem era inversad (ou seja, 48 nm, 532 nm e 410 nm), devido à fotoestabilidade desta amostra particular. </li><li> Defina o limite superior / inferior do Z-stack movendo o estágio do microscópio até a parte superior / inferior das células é um pouco fora de foco. A distância desejada entre cada imagem deve ser mantida constante em 0,125 mm para super-resolução de imagem, como o software de reconstrução tomar este valor como padrão. </li><li> Reconstruir pilhas de imagem usando Softworx (Applied Precision, WA) e analisar usando 6.0 Volocity (Perkin Elmer, MA). </li></ol>

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Enquanto a observação direta de células marcadas com sondas fluorescentes contra LC3 é amplamente aceito como um método padrão para confirmar resposta autofágica 6, imagens 3D quantitativa do mesmo sistema (como temos feito) fornece informações e detalhes sem precedentes sobre o complexo processo de autofagia celular . Em particular, observa-se que centenas (se não milhares) de autophagosomes em células vivas são formadas dentro de 80 min de indução de autofagia. Da mesma forma, observa-se alter...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Arginine Deiminase (ADI)</td>
			<td>DesigneRx</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C5890</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher</td>
			<td>4042</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>S4521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-mouse 555</td>
			<td>Invitrogen</td>
			<td>A21422</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa anti-rabbit 647</td>
			<td>Invitrogen</td>
			<td>A21244</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LysoTracker Red DND-99</td>
			<td>Invitrogen</td>
			<td>L7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Lamp1</td>
			<td>DSHB</td>
			<td>H4A3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>anti-Cadherin</td>
			<td>Cell Signaling</td>
			<td>#3195</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlowFade Gold</td>
			<td>Invitrogen</td>
			<td>S36936</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-d-lysine coated glass bottom plate</td>
			<td>MatTek</td>
			<td>P35GC-1.5-1.4-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>No.1, 22 mm coverslip</td>
			<td>Corning</td>
			<td>#2865-22</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Globe Scientific</td>
			<td>1324G</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

quantitative analysis of autophagy using advanced 3d fluorescence microscopy

O câncer de próstata é a forma principal de tumores malignos entre os homens em os EUA Enquanto a cirurgia acarreta um risco significativo de impotência e incontinência, as abordagens quimioterápicos tradicionais têm falhado. A terapia hormonal é eficaz na fase inicial, mas muitas vezes falha com o eventual desenvolvimento de tumores hormônio-refratário. Nós estávamos interessados ​​no desenvolvimento de terapêuticas dirigidas deficiência metabólica específica de células tumorais. Recentemente, mostrou que as células de tumor da próstata não têm especificamente uma enzima (sintase argininosuccinato ou ASS) envolvida na síntese da arginina, um aminoácido. Esta condição faz com que as células do tumor se tornar dependente de arginina exógena, e eles passam por estresse metabólico quando arginina livre está esgotada pela arginina deiminase (ADI) 1,10. Na verdade, temos demonstrado que as células cancerosas humanas da próstata CWR22 RV1 são efetivamente morto por ADI com apoptose caspase-independente e autopha agressivogy (ou macroautophagy) 1,2,3. A autofagia é um processo evolutivamente conservada que permite que as células de metabolizar proteínas indesejadas pelo colapso lisossômico durante inanição nutricional 4,5. Embora os componentes essenciais desta via são bem caracterizados 6,7,8,9, muitos aspectos do mecanismo molecular não são ainda claras - em particular, o que é o papel de autofagia no-resposta a morte de células de cancro da próstata após o tratamento ADI ? A fim de resolver esta questão, necessário um método experimental para medir o nível ea extensão da resposta autofágica em células - e uma vez que não há marcadores moleculares conhecidos que podem controlar com precisão este processo, optamos por desenvolver uma abordagem baseada em imagens, usando 3D microscopia de fluorescência quantitativa 11,12. Usando CWR22Rv1 células especificamente marcados com sondas fluorescentes para autophagosomes e lisossomos, mostramos que pilhas de imagens 3D adquirido com ou widefield microscopia desconvolução (e mais tarde, com super-resolução, microscopia de iluminação estruturada) pode capturar claramente as fases iniciais de indução autofagia. Com as aplicações de análise de imagem digitais disponíveis comercialmente, podemos facilmente obter informação estatística sobre o número e autophagosome lisossoma, o tamanho, a distribuição, e do grau de co-localização de qualquer célula de imagens. Esta informação permite-nos acompanhar com precisão o progresso da autofagia em células vivas e permite que nossa investigação contínua sobre o papel da autofagia na quimioterapia do cancro.

A sequência da imagem mostrada na Figura 1 mostra as mudanças físicas que ocorrem em CWR22 células durante os primeiros 80 minutos de indução autofagia. Neste e em outros estudos (não mostrado), consistentemente, observada: (1) de deslocamento do núcleo de distância do centro da célula, (2) redução dos pontos de adesão focal, e (3) a translocação geral autofagossomas e lisossomas em direcção ao centro da célula. Além disso, também observamos um pequeno aumento na co-localização (in...

Watch this Scientific Journal Video about Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy at JoVE.com

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Autophagy é um processo ubíquo que permite que as células para se degradarem e reciclar proteínas e organelas. Aplicamos microscopia de fluorescência para visualizar avançada e quantificar o pequeno, mas, alterações físicas essenciais associados com a indução de autofagia, incluindo a formação e distribuição de autofagossomas e lisossomas, e sua fusão em autolysosomes.

Análise Quantitativa de Autophagy usando microscopia de fluorescência avançada 3D

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

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17.8K visualizações.  University of California, Davis. Autophagy é um processo ubíquo que permite que as células para se degradarem e reciclar proteínas e organelas. Aplicamos microscopia de fluorescência para visualizar avançada e quantificar o pequeno, mas, alterações físicas essenciais associados com a indução de autofagia, incluindo a formação e distribuição de autofagossomas e lisossomas, e sua fusão em autolysosomes.

Vídeo: Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizando Único complexos moleculares In Vivo Usando microscopia de fluorescência avançada

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Biologia

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Lapso de tempo de imagens de Mitose Depois de Transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análise rápida e Exploração de microscopia de fluorescência Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

Análise de Autophagy em<em&gt; Penicillium chrysogenum</em&gt; Por a inanição Pads em combinação com microscopia de fluorescência

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Identificação de cinesina-1 Cargas Utilizando microscopia de fluorescência

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source Análise alta Conteúdo Utilizando Automated microscopia de fluorescência Lifetime Imagiologia

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

Imagem latente funcional das fábricas de transcrição Viral usando microscopia de fluorescência 3D

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...