Este trabalho foi financiado pelo NIH concede DA018109 e MH78823 e Neuroscience NIH Grant Blueprint Núcleo P30NS057105 para Washington University.

Preparação cultura <ol><li> Prepare culturas de células dissociadas de ratazana ou rato de células do hipocampo dia pós-natal 0-3 animais 1. Nossos neurônios aderir a uma monocamada subjacente astrócitos, que por sua vez, adere a uma camada de colágeno espalhados em uma lamela espessura número 0. Placa de neurônios a uma densidade de cerca de 500 células por centímetro quadrado. </li><li> Permitir que as culturas a crescer por 10-14 dias para permitir o desenvolvimento e maturação sináptica. Tratam as culturas in vitro no dia 4 com a ARAC antimitóticos para prender um maior crescimento glial. Alimentá-los no dia 5 in vitro com um câmbio médio metade com médias mais Neurobasal B27 suplemento para aumentar a sobrevivência neuronal. </li><li> Durante o período de cultivo, introduzir tratamentos destinados a alterar o número de sinapses funcionalmente silenciada. Nós achamos que uma forma conveniente para silenciar uma grande porcentagem (~ 80%) das sinapses glutamato é um tratamento de 4 horas com 30 mM de potássio. Incubações mais fraco com estímulos despolarizantes induzir silenciamento 2 similar. Estímulo de 4 horas com 50 mM forskolin, um ativador da adenilato ciclase, pode ser usado para despertar a população de terminais de silêncio no início do estudo 3. </li></ol> Visualizando sinapses silenciosas <ol><li> Células deve ter uma densidade relativamente baixa, com processos bem separados neuríticas no dia 14/10 in vitro, de modo a facilitar a visualização dos terminais sinápticos discretos usando microscopia de fluorescência. </li><li> Prepare uma solução estoque de 5 mM FM1-43FX em água destilada. O estoque deve ser mantido no escuro a 4 ° C. Todos os experimentos usando FM1-43FX também deve ser realizado no escuro. </li><li> Desafio das culturas por 2 minutos com 45 mM de cloreto de potássio em uma solução HEPES-salina tamponada contendo 100 mM de cloreto de sódio, cloreto de cálcio 2 mM, 1 mM de cloreto de magnésio, 10 mM de glicose, e 10 mM FM1-43FX. Esta solução também deve conter uma mM e 25 mM NBQX D-APV para bloquear os receptores pós-sinápticos e evitar sinalização recorrentes. Este desafio vai rótulo terminais sinápticos que são capazes de exocitose e endocitose. O tempo de desafio e força são concebidos para causar pelo menos uma rodada de exocitose de vesículas todos os disponíveis nos 4 piscina reciclagem. No entanto, o desafio não é suficiente para causar silenciar adicional de terminais. </li><li> Lavar a cultura para 3 a 5 segundos apenas, usando o mesmo HEPES-salina tamponada sem cloreto de potássio, mas com 500 mM Advasep-7 para remover a tintura inespecífica 5. Note-se que o timing desta etapa é crítica como a aplicação prolongada de Advasep-7 irá resultar na perda de todos rotulagem FM1-43FX. Próxima lavagem em HEPES-salina tamponada sem Advasep-7 durante cinco intervalos de 2 minutos. </li><li> Fix células com paraformaldeído 4% e glutaraldeído 0,2% em PBS, pH 7,4 por 10 minutos. Lave as células, uma vez brevemente com PBS, em seguida, incubar por 15 minutos em solução de bloqueio contendo soro de cabra normal e 4% 0,04% Triton X-100 em PBS. Note-se que FM1-43FX também é muito sensível à quantidade de Triton X-100 usados ​​aqui e em etapas posteriores. De modo geral, FM1-43FX rotulagem é melhor na ausência de qualquer Triton X-100, mas um pouco de detergente é necessário para permeabilizar as células para a imunocoloração subseqüentes. Temos determinada empiricamente que 0,04% Triton X-100 é ideal para exame pré-sináptico das sinapses silenciosas. </li><li> Diluir o anticorpo primário vGluT-1 em solução de bloqueio em uma concentração de 1:2.500. Incubar as células com agitação suave / rotação no diluída vGluT-1 anticorpo por 3 horas à temperatura ambiente. Certifique-se de cobrir pratos sua cultura com papel alumínio para evitar o branqueamento do corante FM1-43FX. </li><li> Lave as células duas vezes em PBS e incubar as células com um anticorpo anti-porco guinea conjugado a um fluoróforo com diferentes características espectrais de FM1-43FX para distinguir as duas manchas. Para nosso experimento, usaremos Alexa 555-conjugados de anticorpos anti porco guinea-a 1:500 em solução de bloqueio. Incubar as células com o anticorpo secundário agitando por 30 minutos em temperatura ambiente. </li><li> Lave as células quatro vezes mais em PBS e depois montar a lamínula sobre uma lâmina de vidro com uma pequena gota de Fluoromount-G. Certifique-se de orientar a lamela para que as células enfrentar o slide. Permitir que os slides para secar durante a noite. </li><li> Agora estamos prontos para adquirir imagens das sinapses ativas e inativas. Prepare um objetivo de óleo 60 x 1,4 com uma abertura numérica em um microscópio confocal de varredura a laser. Coloque um slide no palco, garantindo que o lado lamela está voltada para o objetivo. Depois de focar o slide, encontrar um campo de neurites que não é muito densa. Também é recomendado para evitar a imagem perto da soma das células, como corante FM1-43FX residual pode permanecer lá, mesmo após a lavagem extensiva. Para as nossas imagens experimental, que normalmente ampliar um campo de tamanho 51 51 microns. </li><li> Adquirirz uma pilha de um determinado campo cobrindo uma profundidade de 7,8 microns com 27 0,3 micron-passos. Esta gama é suficiente para cobrir a espessura das neurites na nossa cultura. Para cada passo na z-stack, primeiro exame com a luz laser que excita imunofluorescência do vGluT-1 anticorpos para identificar todas as sinapses glutamatérgicas em um campo do microscópio. Mesmo campo será verificado novamente a cada passo para FM1-43FX fluorescência. Definir a filtragem de densidade neutra e ganhos fotomultiplicador consistente entre todos os lamínulas para um dado experimento, evitando a saturação do sinal. Que normalmente adquirem ≥ 5 campos por condição para cada experimento. </li><li> Usando o software de análise, converter o z-stack em uma imagem de plano único composto. O processo que usamos para essa conversão produz uma imagem com base na intensidade máxima de qualquer plano em cada pixel. A imagem composta é thresholded e vGluT-1 terminais são identificados para análise, sem referência à FM1-43FX mancha. Áreas manchadas, referido como puncta, são marcados como regiões de interesse. Normalmente, selecionar 10 puncta por campo. Em seguida, o canal de FM1-43FX é thresholded e as regiões de interesse são sobrepostos. O software de análise é utilizada para quantificar a intensidade de fluorescência de cada sinal, bem como a área de cada punctum. Um critério absoluto de pixels ou um percentual de pixels critério pode ser usado para distinguir ativos de sinapses inativas. </li></ol> Resultados representante <ol><li> Em nossas culturas, descobrimos que 70-80% das sinapses glutamato, definida por vGluT-1 coloração, apresentam detectáveis ​​FM1-43 coloração 2, 6-8. Em imagens sobrepostas de verde FM1-43 fluorescência e vermelho vGluT-1 coloração isso se reflete como uma abundância de puncta amarelo: sinapses com co-localizada marcadores. Estas são indicadas pelas setas. </li><li> Aproximadamente 20-30% de (vermelho) vGluT-1 puncta positivo deixará de ter (verde) FM1-43 fluorescência acima dos valores basais. Estes são os terminais inativos pré-sináptica, e um exemplo é indicado pela seta. </li><li> A terceira população de sinapses também será observado. Estes são positivas para (verde) FM1-43 fluorescência, mas desprovido de (vermelho) coloração vGluT-1. Estes são ativos sinapses GABAérgicos e um exemplo é indicado pelo asterisco. Estes são excluídos da maioria das nossas análises, mas pode ser explicitamente estudada usando um anticorpo transportador vesicular GABA no lugar do vGluT-1 anticorpos. </li></ol>

<ol>
	<li>Mennerick, S., Que, J., Benz, A., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+and+synaptic+properties+of+hippocampal+neurons+grown+in+microcultures+and+in+mass+cultures.">Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures.</a> J. Neurophysiol. 73, 320-332 (1995).</li><li>Moulder, K. L., Jiang, X., Taylor, A. A., Olney, J. W., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+activity+depresses+synaptic+function+through+an+effect+on+vesicle+priming.">Physiological activity depresses synaptic function through an effect on vesicle priming.</a> J. Neurosci. 26, 6618-6626 (2006).</li><li>Moulder, K. L., Jiang, X., Chang, C., Taylor, A. A., Benz, A. M., Conti, A. C., Muglia, L. J., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+role+for+Ca2+-dependent+adenylyl+cyclases+in+recovery+from+adaptive+presynaptic+silencing.">A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.</a> J. Neurosci. 28, 5159-5168 (2008).</li><li>Sara, Y., Mozhayeva, M. G., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+vesicle+recycling+supports+neurotransmission+during+sustained+stimulation+at+hippocampal+synapses.">Fast vesicle recycling supports neurotransmission during sustained stimulation at hippocampal synapses.</a> J. Neurosci. 22, 1608-1617 (2002).</li><li>Kay, A. R., Alfonso, A., Alford, S., Cline, H. T., Holgado, A. M., Sakmann, B., Snitsarev, V. A., Stricker, T. P., Takahashi, M., Wu, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 809-817 (1999).</li><li>Rosenmund, C., Sigler, A., Augustin, I., Reim, K., Brose, N., Rhee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+control+of+vesicle+priming+and+short-term+plasticity+by+Munc13+isoforms.">Differential control of vesicle priming and short-term plasticity by Munc13 isoforms.</a> Neuron. 33, 411-424 (2002).</li><li>Altrock, W. D., tom Dieck, S., Sokolov, M., Meyer, A. C., Sigler, A., Brakebusch, C., Fassler, R., Richter, K., Boeckers, T. M., Potschka, H., Brandt, C., Loscher, W., Grimberg, D., Dresbach, T., Hempelmann, A., Hassan, H., Balschun, D., Frey, J. U., Brandstatter, J. H., Garner, C. C., Rosenmund, C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+inactivation+of+a+fraction+of+excitatory+synapses+in+mice+deficient+for+the+active+zone+protein+bassoon.">Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.</a> Neuron. 37, 787-800 (2003).</li><li>Ting, J. T., Kelley, B. G., Lambert, T. J., Cook, D. G., Sullivan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+overexpression+depresses+excitatory+transmission+through+both+presynaptic+and+postsynaptic+mechanisms.">Amyloid precursor protein overexpression depresses excitatory transmission through both presynaptic and postsynaptic mechanisms.</a> Proc. Natl. Acad. Sci. USA. 104, 353-348 (2007).</li></ol>

Significado <ol><li> Normalmente as sinapses são pensadas para operar liberando transmissor com uma probabilidade mensurável. Trabalho por nós e os outros torna evidente que algumas sinapses são refratários à liberação de neurotransmissores, apesar de um conjunto completo do transportador do neurotransmissor vesicular e de outros importantes marcadores pré-sináptica 2, 6-8. Nós temos mostrado previamente que a atividade de rede basal em neurônios é fundamental para manter essa população de si...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>FM1-43FX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-35355</td>
<td>A red-shifted FM4-64FX is also available, but has not proven as amenable to assays of presynaptic silencing.</td>
</tr>
<tr>
<td>Advasep-7</td>
<td>&nbsp;</td>
<td>CyDex</td>
<td>AR-0A7-001</td>
<td>&nbsp;</td>
<tr>
<td>vGluT-1</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>AB5905</td>
<td>&nbsp;</td>
<tr>
<td>Alexa 647-conjugated anti-GP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-21450</td>
<td>&nbsp;</td>
<tr>
<td>Fluoromount-G</td>
<td>&nbsp;</td>
<td>Southern Biotechnology Associates</td>
<td>0100-01</td>
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presynaptically silent synapses studied with light microscopy

Synaptic subjaz plasticidade provável capacidade do sistema nervoso de aprender e lembrar e também pode representar uma capacidade de adaptação que impede que outra forma de tornar-se prejudicial insultos neurotóxicos. Temos vindo a estudar uma forma de plasticidade pré-sináptico que é interessante em parte porque ele é expresso como uma comutação digital dentro e fora da capacidade de um terminal pré-sináptico s para liberar vesículas contendo o neurotransmissor glutamato. Aqui demonstramos um protocolo para a visualização do status da atividade de terminais pré-sinápticos em culturas de células dissociadas preparadas do hipocampo de roedores. O método baseia-se na detecção de sinapses ativas usando coloração com uma forma fixable do corante styryl FM1-43, comumente usado para rotular vesículas sinápticas. Este perfil coloração é comparado com imunomarcação dos terminais mesmo com um anticorpo dirigido contra o transportador de glutamato vesicular 1 (vGluT-1), uma mancha projetado para rotular todas as sinapses de glutamato, independentemente do status da ativação. Nós encontramos que os estímulos despolarizantes induzir silenciamento pré-sináptica. A população de sinapses que é silencioso em condições de linha de base pode ser ativado por silenciar elétrica prolongada ou por ativação de vias de sinalização cAMP.

Watch this Scientific Journal Video about Presynaptically Silent Synapses Studied with Light Microscopy at JoVE.com

Presynaptically Silent Synapses Studied with Light Microscopy

Sinapses glutamatérgicas pode mudar de um modo ativo de um modo silencioso. Nós demonstramos que o status de atividade pré-sináptica da cultura dissociada dos neurônios dos roedores é visualizado através de um formulário fixable do FM1-43 corante para visualizar sinapses ativas e imunocoloração com vGluT-1 anticorpos para visualizar todas as sinapses de glutamato.

Sinapses pré-sináptico Silencioso Estudou com Microscopia de Luz

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

presynaptically-silent-synapses-studied-with-light-microscopy

Research

JoVE Journal

Biology

11.4K visualizações.  Washington University School of Medicine. Sinapses glutamatérgicas pode mudar de um modo ativo de um modo silencioso. Nós demonstramos que o status de atividade pré-sináptica da cultura dissociada dos neurônios dos roedores é visualizado através de um formulário fixable do FM1-43 corante para visualizar sinapses ativas e imunocoloração com vGluT-1 anticorpos para visualizar todas as sinapses de glutamato.

Vídeo: Presynaptically Silent Synapses Studied with Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

Preparação de Drosophila Células S2 para microscopia de luz

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Biologia

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Electrosoldável célula visualizado com microscopia de fluorescência

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Resposta celular ao vivo para Estimulação mecânicas estudadas por Microscopia de Força Atômica e Óptica Integrada

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Imagem direta de cálcio ER com Targeted-Esterase Induzida Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Exame<em&gt; Drosophila</em&gt; Larval células terminais traqueais por microscopia de luz

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicamadas de montagem para longo prazo Microscopia de Luz Folha de Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Clonal Rastreamento de Stem hematopoiéticas e células progenitoras Marcado por cinco proteínas fluorescentes usando confocal e microscopia multifotônica

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análise do Gap Junction-dependente Transferência de miARN com 3D-FRAP Microscopia

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Sinapses pré-sináptico Silencioso Estudou com Microscopia de Luz

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