Name: visualizing stromule frequency with fluorescence microscopy
Uploaded: 2016-11-23T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

NOTA: Para este protocolo, temos nos concentrado em ensaio frequência stromule na epiderme de N. deixa benthamiana. Várias linhas transgénicas estáveis foram gerados que pode ser utilizado para este fim, incluindo a 35S PRO: FNRtp: EGFP 13 e NRIP1 Cerulean: 6. Ambas estas linhas mostram a expressão robusta de fluoróforos no estroma do cloroplasto de folhas cultivadas sob uma ampla gama de condições. Alternativamente, fluoróforos segmentados por cloroplastos pode ser transitoriamente expresso em N. benthamiana usando Agrobacterium Transformações 13. Isto é menos do que ideal as linhas transgénicas, dado que as infiltrações de Agrobacterium induzem algumas respostas de defesa basais em N. benthamiana e interacções com Agrobacterium pode alterar a frequência stromule na folha 14, potencialmente complicar a interpretação dos resultados. Finalmente, para visualizar a formação stromule in vitro,cloroplastos pode ser extraído a partir de qualquer espécie de planta, utilizando fluoróforos geneticamente codificados ou um corante fluorescente, tal como descrito na secção 5 abaixo. 9,15 NOTA:. Métodos pormenorizados de cultivo de plantas foram previamente descritos 16 Resumidamente, crescer N. plantas benthamiana em 4 potes &quot;preenchidos com qualquer mistura de solo profissional que fornece uma boa drenagem. Cubra mudas com uma cúpula de plástico transparente para os primeiros 10-14 dias para fornecer um ambiente úmido para a germinação. adicionar qualquer combinação de fertilizantes padrão seguindo as instruções do fabricante para 14- plantas dias de idade. Cultive plantas sob luz branca, usando ~ 100 mol fótons m -2 seg -1 intensidade de luz. As plantas aquáticas regularmente. 1. Preparar as amostras de folhas para visualização NOTA: dinâmica Stromule são afetados por ferindo 8, então a preparação do tecido deve ser realizada imediatamente antes visualizando stromules por microscopia de fluorescência confocal. Idealmente, uma amostra deve ser visualizado com 15 min após remoção a partir da planta. <ol><li> Corte uma pequena secção da folha usando uma lâmina de barbear muito afiada. Sempre utilizar a mesma região da folha de consistência, e ter a certeza de visualizar a mesma superfície (adaxial ou abaxial) em todas as experiências. </li><li> Imediatamente após o corte da secção de folha, submergir a secção de folha em 5 ml de uma seringa sem agulha cheia com água, remover o ar a partir da seringa, e aplicar um vácuo, cobrindo o orifício de seringa com um dedo e puxando o êmbolo. <ol><li> Solte o êmbolo com cuidado para não danificar a secção de folha. Repetir este duas ou três vezes, ou até a maior parte do ar ter sido retirado e a folha parece ser verde profundo. NOTA: Este passo remove o ar na folha que pode interferir com a visualização precisa de stromules. </li></ol></li><li> Adicionar uma gota de água para um slide e colocar a folha nesta secção gota. Em seguida, adicione outro drop de água para a parte superior da folha, e colocar uma lamela de cobertura sobre a folha. Se houver quaisquer bolhas de ar, bata suavemente a lamínula até que sejam removidos. O slide está agora pronto para a microscopia, e deve ser visualizada imediatamente. </li></ol> 2. A visualização Stromules com microscopia de fluorescência confocal <ol><li> Com luz transmitida, o foco sobre um campo de visão perto do centro da secção de folha (de distância a partir das células danificadas pela lâmina de barbear). Usar uma objectiva 20X de modo que muitos cloroplastos podem ser visualizadas simultaneamente. Guardar uma imagem com luz transmitida de modo que tipos de células podem distinguir-se mais tarde, se necessário. </li><li> Alternar a fonte de iluminação para um laser com filtros de excitação e emissão adequados para o fluoróforo ser utilizado. Por exemplo, excita GFP com um laser de 488 nm e recolher emissões entre 500 nm e 525 nm. <ol><li> Usando o software do microscópio, selecione o canal de GFP e clique para ajustar a abertura pinhole a 1 Aiunidade de ry. Escolha de varredura a laser, para começar a visualizar a amostra e, em seguida, clique no canal de GFP para ajustar a intensidade do laser eo ganho de detector para minimizar a potência do laser, enquanto ainda visualizar claramente stromules. Uma vez que essas configurações tenham sido determinadas, mantê-los o mais consistente possível em todos os experimentos. </li></ol></li><li> Prepare uma experiência -stack z que irá recolher uma série de imagens através da epiderme da folha no campo de visão. <ol><li> Para o z -stack, incluir todos os cloroplastos epidérmicas no campo de visão para evitar viés (por exemplo, se os cloroplastos perto da superfície da folha ter mais stromules em condições testadas). </li><li> Captar imagens no intervalo máximo possível, que incluirá todos os stromules mas minimizar o tempo necessário para um -stack z. Por exemplo, se uma unidade de Airy é equivalente a uma secção confocal de focagem é que 2 um de profundidade, tendo uma imagem a cada 2 um é provavelmente ideal. NOTA: O epidermi folhaS é uma superfície irregular, de modo que a profundidade total do -stack z irá variar, dependendo da amostra; a maioria -stacks z exigirá 10-20 imagens, mas pode incluir mais. </li><li> Ajuste a velocidade de digitalização e resolução de imagem conforme necessário para se certificar de que o -stack z é recolhido rapidamente (normalmente dentro de 5-10 min, se possível). Salve o -stack z para análise posterior. </li></ol></li></ol> Processamento 3. Imagem <ol><li> Determinar a freqüência stromule usando qualquer software de análise de imagem. Para este protocolo, recomendamos a utilização ImageJ, que está disponível ao público a partir do National Institutes of Health (http://imagej.nih.gov/ij/), ou o upgrade popular de ImageJ, Fiji (http://fiji.sc / Fiji). </li><li> Mesclar o z -stack em uma única imagem utilizando a projeção de máxima intensidade no software. </li><li> Identificar e contar manualmente todos os cloroplastos na imagem. </li><li> Para cada cloroplasto, determinar visualmente se um ou mais stromulessão vistas estendendo-se desde o cloroplasto na imagem do z mesclada -stack. Para exemplos, ver Figura 1. Observação: Uma vez que as funções biológicas de stromules não são conhecidos, qualquer, cheio de estroma, extensão tubular fina do cloroplasto pode ser considerado como um &quot;stromule&quot;, independentemente do comprimento. Como regra geral, uma extensão cheio de estroma do cloroplasto é um stromule se for menos do que cerca de 1 um de diâmetro ao longo da maioria do seu comprimento 7. Certifique-se que uma pessoa analisa todas as imagens para controlar as diferenças subjetivas para determinar se ou não um cloroplasto tem uma stromule. Um segundo pesquisador deve verificar independentemente as frequências stromule para reduzir ainda mais preconceitos subjetivos. </li></ol> 4. Desenho Experimental e Amostragem NOTA: Stromule frequência é altamente variável entre as folhas, mas vários relatos sugerem que há pouca variação na frequência stromule dentro de uma indivi9,17 dupla folha. <ol><li> Calcular a frequência da folha stromule de um único campo de vista de uma folha. Descarte a folha, e considerar esta uma amostra independente. Não considere campos separados de vista de uma folha como amostras independentes. NOTA: Não há forte consenso sobre a melhor medida essas mudanças na atividade stromule, e até que a função de stromules é mais claramente definida, os investigadores devem continuar a ser criativo em quantificar a dinâmica stromule. Para a comparação entre a literatura, no entanto, as normas comuns de comunicação de dados deve ser utilizado, além de abordagens mais criativas. No mínimo, sempre relatar a frequência de cloroplastos que têm um ou mais stromules. </li><li> Determinar a frequência dos cloroplastos que têm stromules em um campo de visão. Use ImageJ para identificar todos os cloroplastos em um campo de visão. Em seguida, para cada cloroplasto, determinar se o cloroplasto tem formado pelo menos um stromule. frequência stromule relatório como o percentaGE de cloroplastos com um stromule. NOTA: Alguns pesquisadores transformar a percentagem, por exemplo, com a transformação arco-seno, antes de relatar frequências stromule; enquanto esta é uma opção para a análise estatística, o arco-seno de frequência stromule não é um valor intuitivo e não precisa ser relatado no texto principal ou números de um estudo. <ol><li> Como abordagem alternativa, contar o número total de stromules, e dividi-lo pelo número total de cloroplastos. NOTA: Na maioria dos casos, isso irá produzir resultados semelhantes para abordar 4.2; pode superestimar a frequência stromule, no entanto, se um único cloroplasto tem formado muitos stromules. No exemplo mostrado em resultados representativos, por exemplo, vários cloroplastos têm dois ou mais stromules, elevando o número de stromules por cloroplasto para 40/87 (versus uma frequência stromule de 33/87). </li><li> Alternativamente, determinar o comprimento, em vez de frequência stromule stromule. O comprimento pode ser medidoem ImageJ, traçando a stromule com a ferramenta de linha à mão livre. Observação: Uma vez que o papel biológico do stromules permanece desconhecido, não é claro que o comprimento stromule afecta a sua função. Relatam diferenças significativas em comprimento stromule se eles são observados, mas também relatam as frequências stromule (como descrito em 4.2). NOTA: frequência Stromule pode ser altamente variável entre as folhas diferentes sob as mesmas condições de crescimento. Portanto, embora a determinação frequência stromule pode ser demorado, os experimentos devem ser cuidadosamente projetado para incluir amostras de grande tamanho. Usando conjuntos de dados do nosso laboratório, determinou-se que uma amostra de pelo menos 16 plantas por cada tratamento é geralmente suficientemente poderoso para determinar se existe uma diferença estatisticamente significativa de pelo menos uma alteração de 1,5 vezes na frequência stromule entre duas condições (com a norma α = 0,05 e β = 0,80). </li></ol></li><li> A análise estatística dos resultados. <ol><li> Para comparar a mimum stromule frequências de dois tratamentos, utilize o teste t de Welch (também conhecido como o teste t ou t heteroscedástico assumindo variância desigual). NOTA: Este teste não é tão poderoso quanto o teste t de Student convencional, mas o teste t de Welch é vantajoso porque não assume que a variação na distribuição de frequência stromule é semelhante entre os tratamentos. NOTA: Uma vez que a frequência stromule é uma &quot;proporção&quot;, a sua distribuição de amostragem está previsto para ser binomial, em vez do que o normal, o que poderia análise estatística, teoricamente, inclinação, se as amostras são muito baixa ou se a frequência stromule é muito baixo (próximo a 0%) ou muito elevado (perto de 100%). Alguns relatórios têm utilizado arcsine transformações antes da análise estatística, que se destina a transformar uma distribuição binomial a uma distribuição normal, e, assim, satisfazer os requisitos da norma de um teste t de Student ou ANOVA. Na prática, no entanto, umatransformações rcsine têm pouco ou nenhum efeito na análise estatística, e, portanto, não são recomendados. Em vez disso, repetir experiências para aumentar o tamanho da amostra, o que vai aumentar o poder estatístico e reduzir os erros de interpretação de dados. </li><li> Independentemente da abordagem estatística é usado, não se esqueça de informar cuidadosamente a estratégia de amostragem, dimensão da amostra, teste estatístico e valor de p para cada experimento. NOTA: Como com outros campos da biologia, um valor p inferior a 0,05 pode ser considerado &quot;estatisticamente significativa&quot;, embora os pesquisadores certamente deve informar o valor p precisa obtido. Se p é ligeiramente acima de 0,05, aumentando o tamanho da amostra com duas ou três experiências podem contribuir para determinar se ou não a diferença observada é reprodutível e estatisticamente significativa. </li></ol></li></ol> 5. Extrair cloroplastos intactos para Visualize Stromule Dynamics NOTA: Vários métodostêm sido utilizados para isolar os cloroplastos de folhas, incluindo um protocolo ligeiramente diferente num estudo recente sobre a formação in vitro stromule 15. O protocolo detalhado abaixo utiliza um método relativamente simples que não se obter amostras de cloroplastos bioquimicamente puros, mas que em vez disso isolar uma quantidade grande de cloroplastos intactos, 9,18 saudáveis. <ol><li> Prepare tampão de extracção a frio: mM Hepes NaOH 50, sorbitol 330 mM, EDTA 2 mM, MgCl 1,0 mM 2, e MnCl 1,0 mM 2. Ajustar o pH para 6,9 com NaOH e HCl, e refrigerar antes da utilização. Nota: Embora não necessário, utilizando tampões frio e manter as amostras em gelo quando possível irá produzir uma proporção mais elevada de cloroplastos intactos. </li><li> Preparar tampão de isolamento: 50 mM de Hepes NaOH, sorbitol 330 mM, EDTA 2 mM, MnCl 2 1,0 mM, NaCl mM, 1,0 mM de MgCl 2, 10 mM de KCl, e 1,0. Ajustar o pH para 7,6 com NaOH e HCl e refrigerar antes da utilização. </li><li> Se as folhas não sãoexpressando um fluoróforo do estroma geneticamente codificada, diacetato de carboxifluoresceína preparar a solução (CFDA): CFDA solução estoque a 50 mM em sulfóxido de dimetilo (DMSO) (2,3 mg CFDA por 1,0 mL de DMSO). NOTA: A concentração exacta não é crítica. Manter estoque CFDA no escuro em todos os momentos. Armazenar aliquotas de pequenos CFDA 50 mM a -20 ° C. </li><li> Para isolar cloroplastos, remova ~ 5-10 g folhas de várias plantas e lavar brevemente em água fria. transferir imediatamente para 50 ml de tampão de extracção a frio. folhas de moagem, utilizando um liquidificador com vários pulsos curtos. Filtrar através de duas ou três camadas de gaze para remover restos de folhas, dividir os cloroplastos extraídos em dois tubos de centrífuga de 50 ml e centrifugar durante 1 min a 750 x g. </li><li> Descartar o sobrenadante e ressuspender as cloroplastos verdes em tampão de isolamento 10 ml. Centrifugar novamente durante 1 min a 750 xg, descartar o sobrenadante e ressuspender cloroplastos em tampão de isolamento para levar o volume final até 5 ml. </li><li> Transferir 201; l dos cloroplastos para um slide, cubra com uma lamela, e começar a microscopia. NOTA: Os cloroplastos de plantas transgênicas que expressam proteínas fluorescentes segmentadas plastídeo agora está pronto para visualização por microscopia de fluorescência confocal. </li><li> Manchar cloroplastos de plantas que não são expressam proteínas fluorescentes segmentadas plastídeo para visualizar stromules. <ol><li> Adicionam-se 5 ul de estoque 50 mM de CFDA para os 5 ml de cloroplastos-suspensas em tampão de isolamento. Levar a concentração final de 50 uM. </li><li> Permitir cloroplastos para incubar durante 5 min, e, em seguida, transferir uma pequena alíquota (~ 50 jil) a uma lâmina, coberta com uma lamela, e começar a microscopia. </li><li> Use um conjunto de filtros FITC ou GFP. Use um objetivo 20X para visualizar muitos cloroplastos, simultaneamente, ou um objectivo superior para visualizar um único cloroplasto isolado. NOTA: CFDA ficam fluorescentes dentro do estroma dos cloroplastos intactos. </li><li> Se não houver fundo fluorescência forte, lavar o chloroplasts novamente em 10 ml de tampão de isolamento após a 5 minutos de incubação com CFDA, centrifugar durante 1 minuto a 750 xg, descartar o sobrenadante e ressuspender em 5 ml de tampão de isolamento. </li></ol></li></ol>

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The authors have nothing to disclose.

Ao investigar stromules, três fatores importantes devem ser considerados ao longo de: (i) a manipulação do tecido da planta devem ser mantidos a um mínimo absoluto, (ii) o sistema experimental deve ser mantido consistente, e (iii) estratégias de amostragem devem ser cuidadosamente planejado para assegurar robusto, os dados podem ser reproduzidos são analisados. Stromules são notavelmente dinâmica: eles podem se estender e retrair rapidamente diante dos olhos de um observador sob o mi...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

<table><tbody><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>NaOH</td><td>Fischer-Scientific</td><td>S320-1</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>EDTA</td><td>Fischer-Biotech</td><td>BP121</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>221279</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M0250</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>Laser Scanning Confocal Microscope&amp;nbsp;</td><td>Carl Zeiss Inc</td><td>Model: LSM710</td><td></td></tr><tr><td>Carboxyfluorescein diacetate (CFDA)</td><td>Sigma-Aldrich</td><td>21879&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>EMD</td><td>MX1458-6</td><td></td></tr><tr><td>Waring blender</td><td>Waring&amp;nbsp;</td><td>Model: 31BL92</td><td></td></tr><tr><td>Fiji</td><td>fiji.sc</td><td></td><td>Open-source software for analyzing biological images</td></tr></tbody></table>

visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Este protocolo foi usado para visualizar a frequência stromule de dia e à noite nos cotilédones de jovens N. mudas benthamiana. Fatias de uma pilha z foram fundidas em uma única imagem (Figura 1A). Para efeitos visuais, que a imagem foi então Saturado e invertido de modo que o estroma aparece negro (Figura 1B). Os cloroplastos foram marcados quer como não tendo stromules asterisco (verde) ou com pelo menos um stromule (as...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizando Frequency Stromule com microscopia de fluorescência

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

visualizing-stromule-frequency-with-fluorescence-microscopy

Research

JoVE Journal

Biology

8.0K visualizações.  USDA. Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Vídeo: Visualizing Stromule Frequency with Fluorescence Microscopy

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

Monitoramento Espacial Segregação na superfície Colonizar microbianas Populações

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

Genética

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Imagem Lifetime fluorescência de rotores moleculares em células vivas

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Bioengenharia

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Imagens de células vivas de Bacillus subtilis E Streptococcus pneumoniae Usando Microscopia lapso de tempo Automated

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Imunologia e Infecção

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Fluorescência de reflexão interna total de alta taxa de transferência e microscopia de reconstrução óptica estocástica direta usando um chip fotônico

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

Bioquímica

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

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. 
Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic device.

Fluorescência tempo-lapso da completa<em&gt; S. venezuelae</em&gt; Ciclo de Vida Usando um dispositivo micro

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

Ferramentas microfluídicas para sondar interações fúngicas-microbianas no nível celular

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

A quantificação temporal da expressão MAPK induzida em células de levedura Solteiro

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para a rotulagem transcritos em células individuais Escherichia coli para único-molécula fluorescência In Situ as experiências de hibridação

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Visualizando Frequency Stromule com microscopia de fluorescência

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