Gostaríamos de agradecer Microsystems Technology Laboratories no MIT por nos permitir filme parte deste vídeo na instalação de sala limpa.

Preparação 1. Crie uma Máscara <blockquote> Usando um software CAD, design do canal de alta resolução de impressão em uma transparência. Esta será a &quot;máscara&quot;. </blockquote> Na sala limpa: 2. Limpe e coza a bolacha Primeiro, o wafer esguicho com acetona, em seguida, rapidamente, com metanol, em seguida, com Isopropanol. Por fim, seque o wafer usando nitrogênio. Asse o wafer no forno (130 ° C) por 5 min. 3. Revestimento do wafer Coloque a bolacha no centro da máquina de spin-coating. Despeje fotorresiste (SU-8) da garrafa para o wafer. Permitir o fluxo de SU-8 e relaxar por ~ 10 s. Ligue o spin-coater e rampa a sua velocidade até 0-500 rpm por 5 s; manter a 500 rpm por 10 s; rampa até a velocidade final de mais de 10 s e manter a velocidade final de 30 s. A velocidade final depende da espessura do revestimento alvo e os SU-8 usada. Os detalhes podem ser encontrados em <a href="http://www.microchem.com/">http://www.microchem.com/</a> 4. Soft-bake Após o revestimento do wafer, cozê-lo primeiro em 65 ° C e depois a 95 ° C. O tempo de cozimento varia de acordo com a espessura alvo e tipo de fotorresiste utilizado. Então, deixe o wafer sentar-se à temperatura ambiente por pelo menos 5 min. 5. Exposição Colocar a máscara em cima do wafer e expor a hóstia à luz UV durante o tempo recomendado no manual do SU-8. 6. Pós-exposição bake Asse o wafer de 65 ° C e 95 ° C, em seguida, após o SU-8 manual de instruções. 7. Desenvolvimento do wafer para obter o &quot;master&quot; (molde) Prepare um copo cheio do desenvolvedor (PMMA). Mergulhar a bolacha no copo, enquanto muito gentilmente oscilando o copo até que a parte não exposta do fotorresiste é lavado. Em nosso laboratório: 8. Prepare PDMS e derramá-lo sobre o wafer Misture o PDMS com seu agente de cura em razão de 10:1 em um copo. Mexa e misture de forma homogênea: isso vai gerar muitas bolhas e fazer a mistura olhar opaco. Despeje a mistura sobre o &quot;mestre&quot;. 9. De-bolha no vácuo da câmara Para remover as bolhas, colocou a mistura mestre e PDMS que está cobrindo-o em uma câmara de vácuo até que todas as bolhas se foram. 10. Assando no forno Leve ao forno por, pelo menos, 12 horas em estufa a 65 ° C para endurecer o PDMS. 11. Furos Retire a PDMS do mestre e furos para entradas e saídas dos canais. Em salas limpas (não mostrado) 12. Plasma de ligação Canais são ligados a uma lâmina de vidro após o tratamento tanto a camada de PDMS e lâmina de vidro com plasma de oxigênio por 1 min. Experimentos: Exp # 1: Investigar a resposta quimiotática de micróbios marinhos para micro-escala camadas de nutrientes 1) Configurando o experimento <ol><li> Adicionar organismos e substratos para seringas de vidro </li><li> Lugar canal microfluídicos para o palco microscópio e anexar tubulação para entradas e saídas apropriadas </li><li> Conecte a tubulação de resíduos do reservatório. Certifique-se de tubulação é totalmente submersos no fluido no reservatório de resíduos para evitar oscilações de pressão </li><li> Seringas lugar em bomba de seringa e conecte-se válvulas e tubulação </li><li> Configurar microscópio: condições de iluminação, ampliação, etc </li><li> Concentre-se em posição apropriada no canal </li><li> De-bolha canal usando seringa maior cheia com água do mar artificial </li><li> Defina a taxa de fluxo apropriado em bomba de seringa. Neste caso, 2 ml / min, que corresponde a uma velocidade de fluxo médio de 220 ​​mm s -1 no canal </li></ol> 2) A execução do experimento <ol><li> Iniciar bomba de seringa para estabelecer um gradiente de nutrientes no canal </li><li> Quando o fluxo se estabilizou, e uma banda de nutrientes tem desenvolvido, interromper o fluxo da bomba de seringa e começar a gravar o tempo a partir deste ponto </li><li> Banda de nutrientes começa a se difundir lateralmente </li><li> Em intervalos regulares de tempo, use o software de análise de imagem para gravar sequências de frames para criar &quot;filmes&quot; </li><li> Discriminar os organismos de natação, tendo tempo de diferença entre duas imagens quadros subseqüentes, de modo que somente objetos em movimento agora são visualizadas, permitindo-nos diferenciar células móveis de não-movimento de partículas e de ruído de fundo </li><li> Fazer filmes para determinar posições de células do canal com referência ao pOSIÇÃO do patch de nutrientes </li><li> Gravar filmes em intervalos regulares por 10-20 min para analisar as posições e padrões de natação de organismos </li><li> Usando o software de análise de imagem para sobrepor as posições de organismos em diferentes quadros (atribuindo a cada pixel a intensidade de luz máxima registrada naquele pixel ao longo da duração do filme), podemos obter informações trajetória para as células de natação </li></ol> Exp # 2: Investigar os efeitos de cisalhamento em bactérias marinhas nadando em um vortexZ <ol><li> Usando a geometria canal diferente, podemos observar o comportamento das bactérias nadando em um vórtice em diferentes taxas de cisalhamento </li></ol>

<ol>
	<li>Azam, F., Malfatti, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+structuring+of+marine+ecosystems.">Microbial structuring of marine ecosystems.</a> Nature Reviews Microbiology. 5, 782-791 (2007).</li><li>Blackburn, N., Azam, F., Hagstrom, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+explicit+simulations+of+a+microbial+food+web.+.">Spatially explicit simulations of a microbial food web. .</a> Limnology and Oceanography. 42, 613-622 (1997).</li><li>Blackburn, N., Fenchel, T., Mitchell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nutrient+patches+in+plankton+habitats+shown+by+chemotactic+bacteria.">Microscale nutrient patches in plankton habitats shown by chemotactic bacteria.</a> Science. 282, 2254-2256 (1998).</li><li>Keymer, J. E., Galajda, P., Muldoon, C., Park, S., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+metapopulations+in+nanofabricated+landscapes.">Bacterial metapopulations in nanofabricated landscapes.</a> Proceedings of the National Academy of Science. 103, 17290-17295 (2006).</li><li>Mao, H., Cremer, P. S., Manson, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+versatile+microfluidic+assay+for+bacterial+chemotaxis.">A sensitive, versatile microfluidic assay for bacterial chemotaxis.</a> Proceedings of the National Academy of Science. 100, 5449-5454 (2003).</li><li>Marcos, R., Stocker, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microorganisms+in+vortices:+a+microfluidic+setup.">Microorganisms in vortices: a microfluidic setup.</a> Limnology and Oceanography: Methods. 4, 392-398 (2006).</li><li>Park, S., Wolanin, P. M., Yuzbahyan, E. A., Lin, H., Darnton, N. C., Stock, J. B., Silberzan, P., Austin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+topology+on+bacterial+social+interaction.">Influence of topology on bacterial social interaction.</a> Proceedings of the National Academy of Sciences. 100, 13910-13915 (2003).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annual Review of Biomedical Engineering. 3, 335-373 (2001).</li></ol>

Uma compreensão de como micróbios marinhos interagem com seus química local e ambiente físico é imprescindível para uma percepção mais completa e precisa do papel de microorganismos planctônicos na nutrientes e carbono oceanos ciclos (Azam e Malfatti 2007). No entanto, devido à pequena escala (mm &lt;) sobre o qual muitas interações microbiana importante ter lugar, as limitações técnicas impediram exames detalhados do comportamento microbiano na paisagem bio-físico-químicas heterogêneas previsto para ser experimentado por ...

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<td>PDMS, Sylgard 184</td>
<td>Silicone Elastomer Kit</td>
<td>Dow Corning, Midland, MI, USA</td>
<td>&nbsp;</td>
<td>http://www.ellsworth.com/sylgard.html</td>
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<td>SU8-2100</td>
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<td>MicroChem, Newton, MA, USA</td>
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<td>Nikon Eclipse TE2000-E inverted microscope</td>
<td>Microscope</td>
<td>Nikon, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PEEK tubing (0.762 mm ID, 1.59 mm OD)</td>
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<td>&nbsp;</td>
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<td>Syringes (Luer-Lok Tip)</td>
<td>Tool</td>
<td>BD, Franklin Lakes, NJ, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fitting Part P-704-01</td>
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<td>Upchurch Scientific, Oak Harbor, WA, USA</td>
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<td>To connect tubing to Luer-Lok Tip Syringes</td>
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<td>Harvard Apparatus, Holliston, MA, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Equipment</td>
<td>Cooke, Romulus, MI, USA</td>
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chemotactic response of marine micro-organisms to micro-scale nutrient layers

O grau em que os micróbios podem explorar planktonic manchas de recursos microescala terá implicações consideráveis ​​para trophodynamics oceânicas e de fluxo biogeoquímicos. No entanto, para tirar proveito de patches de nutrientes no oceano, os micróbios natação deve superar as influências de forças físicas incluindo a difusão molecular e turbulenta de cisalhamento, o que limitará a disponibilidade de patches e a capacidade das bactérias para localizá-los. Até recentemente, as limitações metodológicas têm impedido exames direta do comportamento microbiano dentro de habitats irregular e condições realistas de pequena escala de fluxo. Assim, muito do nosso conhecimento atual em relação ao comportamento microbiano no oceano foi obtida a partir de previsões teóricas. Para obter novas informações sobre o comportamento de forrageamento microbiana no oceano temos aplicado suave técnicas de fabricação litográfica para desenvolver dois dispositivos microfluídicos, que temos usado para criar (i) micro nutrientes manchas com dimensões e características difusivos relevantes para processos oceânicos e (ii) microescala vórtices, com taxas de cisalhamento correspondente aos esperados no oceano. Estes dispositivos microfluídicos têm permitido um primeiro exame direto de natação microbiana e comportamento quimiotáticos dentro de um seascape heterogêneo e dinâmico. O uso combinado de epifluorescência e microscopia de contraste de fase permitem exames diretos das dimensões físicas e as características difusivo de patches de nutrientes, enquanto observa a resposta agregativa em nível de população, além do comportamento de natação de micróbios individuais. Esses experimentos revelaram que algumas espécies de fitoplâncton, bactérias heterotróficas e protistas phagotrophic são peritos em localizar e explorar difusão manchas de recursos microescala em prazos muito curtos. Também mostramos que até moderada taxas de cisalhamento, bactérias marinhas são capazes de lutar contra o fluxo e nadar através de seu ambiente de sua própria vontade. No entanto, além de um nível de cisalhamento alto limiar, bactérias estão alinhados no fluxo de cisalhamento e são menos capazes de nadar sem perturbação do fluxo. Microfluídica representa uma nova abordagem e barato para estudar a ecologia microbiana aquática, e devido a sua aptidão para a criação de campos de fluxo com precisão realista e gradientes de substrato em microescala, é idealmente aplicável aos exames de comportamento microbiana nas menores escalas de interação. Assim, sugerimos que a microfluídica representa uma valiosa ferramenta para a obtenção de uma melhor compreensão da ecologia de microrganismos no oceano.

Watch this Scientific Journal Video about Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers at JoVE.com

Meiosis  | Biology Video Collection

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

A fabricação de canais microfluídicos ea sua aplicação em experimentos para estudar o comportamento de forrageamento quimiotática de micróbios marinhos de uma seascape irregular de nutrientes eo comportamento de natação de bactérias dentro do fluxo de cisalhamento são descritos.

Resposta quimiotática de microorganismos marinhos para Micro-Escala de Camadas de nutrientes

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

chemotactic-response-marine-micro-organisms-to-micro-scale-nutrient

Research

JoVE Journal

Biology

13.2K visualizações.  MIT - Massachusetts Institute of Technology. A fabricação de canais microfluídicos ea sua aplicação em experimentos para estudar o comportamento de forrageamento quimiotática de micróbios marinhos de uma seascape irregular de nutrientes eo comportamento de natação de bactérias dentro do fluxo de cisalhamento são descritos.

Vídeo: Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth.  Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

Camadas de Simbiose - Visualizando a térmita comunidade microbiana do intestino posterior

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different ...

Biologia

Micro-scale Engineering for Cell Biology

Micro-escala de Engenharia de Biologia Celular

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

Em Imaging vivo do Deep camadas corticais usando um Microprisma

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Usando a resposta optocinético para estudar a função visual de zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantificação de γH2AX Focos em resposta à radiação ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Measurement of Survival Time in<em&gt; Brachionus</em&gt; Rotifers: Sincronização de condições maternas

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hidroponia: um sistema versátil para estudar nutrientes de alocação e de plantas As respostas a disponibilidade de nutrientes e exposição a elementos tóxicos

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. 
Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.

We demonstrate the use of various microscopy methods that are useful in observing the calcification of a tubeworm, Hydroides elegans, as well as locating and characterizing the first calcified material. Live microscopy and electron microscopy are used together to provide functional and material information that are important in studying biomineralization.

Caracterização de calcificação eventos usando o Live Óptica e Microscopia Eletrônica de técnicas em um tubeworm Marinha

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Detecção de activados por ligandos L acoplado a proteína do receptor de internalização por Microscopia Confocal

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Avaliação de Dictyostelium discoideum resposta à estimulação mecânica aguda

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...