Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

Esta pesquisa foi apoiada pela National Science Foundation através de subvenções PHY-1205494 e MCB-1413134 (EL) e pela 2017R1D1A1B03035671 de concessão da Fundação de pesquisa nacional da Coreia (KSL).

O protocolo abaixo usa WormSpa5, um dispositivo microfluidic descritas anteriormente para a imagem latente longitudinal de vermes. Fabricação de WormSpa (começando com arquivos de CAD que podem ser obtidos os autores mediante solicitação) é simples, mas requer alguma experiência. Na maioria dos casos, fabricação pode prontamente ser feita por uma instalação de núcleo ou por uma empresa comercial que fornece esses serviços. Quando o dispositivo de fabricação, certifique-se de especificar que a altura das características 50 µm.
1. experimental Setup
<ol><li>Monte o dispositivo microfluidic em um microscópio de fluorescência invertido com um palco motorizado.</li>
	<li>Coloque um agitador orbital perto do microscópio, mas certifique-se que a vibração do agitador não afeta diretamente o microscópio. Por exemplo, colocar o agitador em uma prateleira fixada em uma parede que faz sem contato com a tabela de óptica.</li>
	<li>Coloca uma bomba de seringa no agitador orbital. Fita da bomba para o agitador para que ele não jiggle agitando.</li>
</ol>2. preparação do ambiente microfluídicos
Nota: Durante o experimento, quatro seringas estão ligadas ao dispositivo microfluidic através de tubos de seringa, conforme descrito na Figura 1. Este passo descreve a preparação destas seringas. A não ser mencionado, caso contrário, manter os tubos de seringa tão curtos quanto possível sem torná-los tenso.
<ol><li>Prepare uma seringa de saída e um tubo de seringa. Esta seringa (S1 na Figura 1) será mais tarde conectados à tomada do dispositivo através do tubo de seringa e usado para armazenamento temporário de líquido saindo do dispositivo.<ol>
<li>Fazer uma agulha de adaptador, removendo as peças de plástico de uma ponta de seringa de 20 x 1/2 polegada, mantendo apenas a agulha (parte metálica). Fazê-lo, mantendo as peças de plástico e de metal com um alicate e separá-los. Resíduo plástico restante na agulha pode ser queimado com um bico de Bunsen.</li>
		<li>Dobre a agulha de acordo com a geometria da instalação experimental, mantendo suas duas extremidades com um alicate. No projeto descrito aqui, dobrar a agulha em seu meio para um ângulo de ~ 110° é mais conveniente.</li>
		<li>Conecte a agulha do adaptador a meio caminho do tubo. A outra metade será mais tarde conectada no dispositivo. O tubo da seringa deve ser compatível com uma ponta de seringa de 20 G sem um vazamento (por exemplo, um tubo com diâmetro interno de 0,86 mm e diâmetro externo de 1,32 mm). O comprimento do tubo recomendado é ~ 50 cm.</li>
		<li>Conecte uma seringa luer-lock de 10 mL para a outra extremidade (aberta) do tubo seringa através de uma ponta de seringa de 20 x 1/2 polegada.</li>
	</ol>
</li>
	<li>Preparar amortecedor-entrada seringas e tubos de seringa. Dentre essas seringas (S2 na Figura 1) é usado como um reservatório para o líquido que entra no aparelho e para controlar o fluxo de injeção. A outra seringa (S3 na Figura 1) é necessária para troca de amortecedor, conforme explicado no passo 3.2.<ol>
<li>Conectar uma seringa de 10ml luer-lock (S2) diretamente a uma válvula de 3 vias e conectar outra seringa (S3) para a mesma válvula através de um tubo de seringa (Figura 1): S3 conectar-se ao tubo através de uma ponta de seringa e ligar o tubo para a válvula através de uma outra ponta de seringa. Nota: Não importa a ordem em que esses materiais estão conectados.</li>
		<li>Conecte um tubo de seringa à porta restante da válvula de 3 vias. Prepare outra agulha adaptador como na etapa 2.1.2 e conecte a outra extremidade (aberta) do tubo seringa a agulha na metade do caminho.</li>
		<li>Conjunto da válvula, tal que o tubo de seringa na etapa 2.2.2 e S2 estão ligados enquanto S3 é fechado (Figura 1).</li>
	</ol>
</li>
	<li>Prepare uma seringa de worm-entrada e um tubo de seringa. Esta seringa (S4 na Figura 1) é usado para carregar vermes no dispositivo.<ol>
<li>Conecte um tubo de seringa longa uma seringa de 10ml luer-lock (S4). Determinar o comprimento deste tubo do volume do fluido que será usado para o carregamento; por exemplo, 100 cm de tubo suporta mais de 2 mL de líquido (consulte também o passo 4.2).</li>
		<li>Prepare outra agulha adaptador como na etapa 2.1.2 e ligar um meia da agulha para a outra extremidade (aberta) do tubo seringa.</li>
	</ol>
</li>
	<li>Lave todas as seringas e tubos com S-médio12 duas vezes. Encha as seringas e os tubos com S-meio, tendo o cuidado de remover todas as bolhas de ar.</li>
	<li>Conecte o tubo de saída da seringa à saída do dispositivo.<ol>
<li>Conecte a agulha do adaptador na extremidade do tubo seringa à porta de saída.</li>
		<li>Injete um pequeno volume de S médio através da saída para encher o dispositivo, até um pouco S médio saia tanto a entrada da reserva e a entrada de verme.</li>
	</ol>
</li>
	<li>Conecte o tubo de seringa de tampão-entrada à porta de entrada de buffer do dispositivo, enquanto obstruindo a entrada de verme com um pino (pino de passador de aço inoxidável com um diâmetro de 1/32 de polegada).</li>
	<li>Injete um fluxo constante de buffer do dispositivo. Carregar a seringa de tampão-entrada S2 para uma bomba de seringa13e conjunto da bomba tal que a média em S2 é injectada continuamente o dispositivo com um caudal de 3 µ l/min.</li>
</ol>3. carregar as bactérias para o dispositivo Microfluidic
<ol><li>Prepare-se Escherichia coli OP50 suspendido em S-médio12.<ol>
<li>Na sequência de um protocolo padrão, inocular um balão de 250 mL com 50 mL de LB e uma única colônia e incubar durante uma noite a 37 ° C.</li>
		<li>Centrifugar a cultura durante a noite a 4000 rpm por 10 minutos e resuspenda o pellet em 30 mL de meio-de-S.</li>
		<li>Filtre a suspensão através de um filtro de seringa de 5 µm. Medir a concentração bacteriana por sua densidade óptica em 600 nm (OD600) e ajustar a concentração de uma OD600 de 3. Esta alta densidade é necessária para manter os vermes bem alimentados.</li>
	</ol>
</li>
	<li>Troca o buffer no dispositivo com a suspensão de OP50.<ol>
<li>Prepare uma seringa limpa (S5) preenchida com a suspensão de OP50. Segurar a seringa verticalmente e bata-o várias vezes para recolher todas as bolhas de ar no topo.</li>
		<li>Gire a válvula de 3 vias de seringas de tampão-entrada para fechar a conexão para o dispositivo e conecte as duas seringas, S2 e S3. Desengate o S2 da bomba de seringa.</li>
		<li>Desconecte a válvula-S2 e S5 se conectem a válvula. Expulsar todo o ar coletado no topo da S5 S3 através da válvula até um pouco de buffer OP50 entra S3. Ao fazer isso, certifique-se de que nenhuma bolha de ar permanece em S5 ou a válvula.</li>
		<li>Gire a válvula de 3 vias para a posição original para fechar a conexão entre S3 e S5 e conectar o dispositivo a S5. S5 de carga sobre a bomba de seringa. Ligue o agitador orbital que prende a bomba. Defina a velocidade de agitação para cerca de 200 rpm.</li>
		<li>Defina a taxa de fluxo ser 100 µ l/min. O tempo que leva para o novo buffer chegar para os vermes no dispositivo depende do comprimento do tubo seringa reserva-entrada (cerca de 5 minutos com a tubagem descrito acima). Uma maior taxa de fluxo pode ser empregada se uma troca de amortecedor mais rápida é necessária.</li>
		<li>Uma vez que o buffer OP50 se espalha por todo o dispositivo, defina a taxa de fluxo para 3 µ l/min.</li>
	</ol>
</li>
</ol>4. carregar vermes no dispositivo Microfluidic
<ol><li>Prepare um pequena ligação baixa microcentrifuga tubo (650 µ l) e preenchê-lo com 100 µ l de suspensão de OP50. Transferência em torno de 20-30 jovens de idade-sincronizado pegar germes adultos (46 horas após a detenção larval de L1 a 25 ° C) de uma placa de ágar NGM (meio de crescimento nemátodes) ao tubo por pegá-los um por um com um verme. Idade-sincronização pode ser feito na sequência de um protocolo padrão12.</li>
	<li>Preencher o worm-entrada da seringa e seringa do tubo (S4 na Figura 1) com a suspensão de OP50. Injete ~ 500 µ l do fluido dentro do tubo de microcentrifugadora com vermes. Desenhe a suspensão com vermes no tubo de seringa de worm-entrada. Certifique-se que o tubo da seringa é longa o suficiente (> 1 m) e que vermes desenhadas fiquem no tubo de seringa e não fazem todo o caminho até a seringa S4.</li>
	<li>Conecte o S4-entrada do worm. Injete o dispositivo microfluidic todos os vermes no tubo de seringa de worm-entrada. Desconecte o S4 e o tubo de seringa. Conecte a entrada de verme com um alfinete.</li>
	<li>Desencaixe a seringa de tampão-entrada S2 da bomba mecânica ( Figura 1) e controlar manualmente S1 e S2 para empurrar todos os vermes em canais separados. Pressionar S2 moverá vermes os canais, enquanto pressionar S1 irá movê-los na direção inversa. Uma vez que um canal é carregado, o verme em cada canal irá bloquear a entrada de outros worms.</li>
	<li>Deixe vermes ajustar ao novo ambiente, por 2-3 horas.</li>
</ol>5. criação de um protocolo de imagem
<ol><li>Encontrar todos os vermes que localizam-se firmemente no dispositivo e definir uma posição de imagem para cada um no software de aquisição de imagem que controla seu microscópio. Observe que em alguns casos (por exemplo, quando se deseja uma ampliação maior, ou quando utilizar câmeras com um sensor CCD menor) múltiplas posições de imagem são necessárias para cada canal. Nota: Enquanto isso pode ser feito manualmente, alguns pacotes de software de aquisição podem carregar um arquivo de texto que lista as posições onde as imagens devem ser tomadas. Desde que estas posições são ordenadas regularmente em WormSpa, um usuário pode escrever um pequeno script que cria automaticamente uma lista desse tipo (por exemplo, em Python ou software comercial). O formato preciso desse script depende do formato de arquivo esperado pelo software de aquisição (veja um exemplo no Material complementar 1).</li>
	<li>Defina a frequência necessária da imagem latente de lapso de tempo. Por exemplo, imagem latente da resposta de gene imune à infecção é feito com uma frequência de 10 minutos por quadro. Certifique-se de que todos os canais necessários (por exemplo, brilhante-campo e a fluorescência de GFP) são adquiridos em cada ponto de tempo.</li>
</ol>6. host resposta à infecção de Pseudomonas
Nota: Este passo é específico para estudar interações patógeno-hospedeiro. Alternativamente, um pode preparar um buffer que contém outras pistas ambientais de interesse (estresses bióticos e abióticos, drogas, sinalização moléculas, etc.).
<ol><li>Prepare-se Pseudomonas aeruginosa PA14 suspendido no SK-médio14.<ol>
<li>Inocular um balão de 250 mL com 50 mL de LB e uma única colônia e incubar durante uma noite a 37 ° C.</li>
		<li>Inocular o outro balão de 250 mL com 50 mL de LB e uma alíquota da cultura e incubar durante uma noite a 37 ° C.</li>
		<li>Centrifugar a cultura durante a noite a 4000 rpm por 10 minutos e resuspenda o pellet em 10 mL de SK-médio. Medir a concentração bacteriana e ajustar a concentração de OD600 = 4.</li>
		<li>A suspensão de transferência para um balão limpo e incubar a 37 ° C por 24 horas e a 25 ° C, durante 24 horas e agitando. Esta etapa imita a etapa de incubação-placa na norma de ensaio14a matar.</li>
		<li>Filtre a suspensão através de um filtro de seringa de 5 µm. Isso é para evitar agregados bacterianos de obstruir o dispositivo.</li>
	</ol>
</li>
	<li>Substitua a suspensão PA14, seguindo o mesmo procedimento como no passo 3.2 a suspensão OP50 no dispositivo. Manter a imagem por 10 horas.</li>
</ol>

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S., Levine, E., Biron, 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+scalable+method+for+automatically+measuring+pharyngeal+pumping+in+C.+elegans.">A scalable method for automatically measuring pharyngeal pumping in C. elegans.</a> J Neurosci Methods. 274, 172-178 (2016).</li><li>Scholz, M., Dinner, A. R., Levine, E., Biron, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+feeding+dynamics+arise+from+the+need+for+information+and+energy.">Stochastic feeding dynamics arise from the need for information and energy.</a> Proc Natl Acad Sci U S A. 114 (35), 9261-9266 (2017).</li></ol>

Os autores não têm nada para divulgar.

Microfluidic ferramentas fornecem vários benefícios no estudo de vermes. Imaging em um PDMS dispositivo oferece maior qualidade de imagem em comparação com uma placa de ágar padrão NGM. Várias imagens podem ser tomadas a partir de um único verme, em contraste com os métodos tradicionais em que os animais são colhidos a partir da placa e montados sobre uma lâmina de microscópio para a imagem latente. Além disso, o microambiente no qual residem vermes pode ser mantido constante ou modulado conforme desejado, p...

Mudanças nas condições ambientais podem levar a ativação de programas genéticos, acompanhado por indução e repressão da expressão de genes específicos1,2. Essas alterações cinéticas podem ser variável entre tecidos nos mesmos animais e entre animais diferentes. Estudos de tais programas genéticos, portanto, chamar para métodos que permitem a imagem longitudinal de animais individuais e fornecem controle dinâmico preciso das condições ambientais.
Nos últimos anos, microfabricated fluídico dispositivos têm sido usados para estudar muitos aspectos da resposta e comportamento em pequenos animais, incluindo vermes, moscas, água ursos e mais3,4,5,6, 7. As aplicações incluem, por exemplo, fenotipagem profunda, optogenetic gravação de atividade neuronal em resposta a estímulos químicos e monitoramento de comportamentos motor, tais como locomoção e bombeamento8,9,10 , 11.
Abordagens baseadas em microfluidic segurar muitas propriedades que poderiam beneficiar a longo prazo de imagem longitudinal da resposta a sinais ambientais, incluindo controle dinâmico preciso do microambiente local, design flexível que permite a manutenção de cada animal em quartos separados e atributos favoráveis para a imagem latente. No entanto, manter animais em uma câmara de microfluidic por um longo tempo com o mínimo impacto adverso sobre seus seres bem é um desafio que requer uma atenção especial no projeto do dispositivo microfluidic, bem como na execução do experimento.
Aqui vamos mostrar o uso de WormSpa, um dispositivo microfluidic para imagem longitudinal de Caenorhabditis elegans. 5 vermes individuais estão confinados em câmaras. Um baixo fluxo constante de suspensão bacteriana e líquido garante que vermes são bem alimentados e suficientemente ativo para manter a boa saúde e aliviar o estresse, e a estrutura das câmaras permite vermes para pôr ovos. A simplicidade do projeto e operação de WormSpa devem permitir que os investigadores com nenhuma experiência anterior em microfluídica para incorporar este dispositivo em seus próprios planos de pesquisa.

<table><tbody><tr><td>WormSpa</td><td>N/A</td><td>N/A</td><td>The CAD file for WormSpa is available from the Levine lab.</td></tr><tr><td>Compound Microscope</td><td>Zeiss</td><td>AxioObserver Z1</td><td>An inverted fluorescence microscope with a motorized stage</td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems</td><td>NE-501</td><td></td></tr><tr><td>Tubing</td><td>SCI Scientific Commodities Inc.</td><td>BB31695-PE/5</td><td>0.034&amp;rdquo; (0.86 mm) I.D. x 0.052&amp;rdquo; (1.32 mm) O.D</td></tr><tr><td>Syringe Tip</td><td>CMLsupply</td><td>901-20-050</td><td>20 Gauge x 1/2&amp;rdquo; blunt tip stainless steel canula</td></tr><tr><td>Syringe Filter</td><td>PALL</td><td>4650</td><td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td></tr><tr><td>Syringe</td><td>Qosina</td><td>C3307</td><td>10 mL Male Luer Lock Syringe</td></tr><tr><td>3 Way Valve</td><td>ColeParmer</td><td>FF-30600-23</td><td>Large-bore 3-way, male-lock, stopcocks, 10/pack, Non-sterile</td></tr><tr><td>Dowel Pin</td><td>McMaster-Carr</td><td>90145A317</td><td>18-8 Stainless Steel Dowel Pins (1/32&quot; Dia. x 1/2&quot; Lg.)</td></tr><tr><td>Low Binding Microcentrifuge Tube</td><td>Corning</td><td>CL S3206</td><td>0.65 mL low binding snap cap microcentrifuge tube</td></tr></tbody></table>

a microfluidic platform for longitudinal imaging in caenorhabditis elegans

Na última década, microfluidic técnicas foram aplicadas para estudar pequenos animais, incluindo o nematódeo Caenorhabditis elegans, em provaram útil como uma plataforma de imagem ao vivo conveniente, fornecendo recursos para o controle preciso de condições experimentais em tempo real. Neste artigo, vamos mostrar imagens ao vivo de vermes individuais, empregando o WormSpa, um dispositivo microfluidic personalizado anteriormente publicada. No dispositivo, vários vermes limitam-se individualmente para separar câmaras, permitindo multiplexada vigilância longitudinal de vários processos biológicos. Para ilustrar o recurso, realizamos experimentos de prova de princípio em que minhocas foram infectadas no dispositivo com bactérias patogênicas e a dinâmica da expressão de genes de resposta imune e postura de ovos foram monitorada continuamente no indivíduo animais. O design simples e operação deste dispositivo torná-lo adequado para usuários sem experiência anterior com experimentos microfluidic-baseado. Propomos que esta abordagem será útil para muitos pesquisadores interessados nas observações longitudinais de processos biológicos, sob condições bem definidas.

Idade-sincronizado em vermes de adultos jovens (46 horas pós prisão larval de L1 a 25 ° C)12 foram carregados no dispositivo, conforme descrito no protocolo. Individualmente, os vermes estavam localizados em canais separados, permitindo a medição longitudinal da resposta dos animais ao patógeno. Quando a experiência for bem sucedida, a maioria dos vermes permanecem em seus canais para a duração do experimento. Neste caso, imagens de worms individuais são ...

Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

Uma plataforma Microfluidic para Imaging Longitudinal em Caenorhabditis elegans

Neste artigo, vamos mostrar imagens ao vivo de vermes individuais empregando um dispositivo microfluidic personalizado. No dispositivo, vários vermes limitam-se individualmente para separar câmaras, permitindo multiplexada vigilância longitudinal de vários processos biológicos.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

a-microfluidic-platform-for-longitudinal-imaging-caenorhabditis

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

6.4K visualizações.  Kongju National University. Neste artigo, vamos mostrar imagens ao vivo de vermes individuais empregando um dispositivo microfluidic personalizado. No dispositivo, vários vermes limitam-se individualmente para separar câmaras, permitindo multiplexada vigilância longitudinal de vários processos biológicos.

Vídeo: A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos microfluídicos para<em&gt; In vivo</em&gt; Imaging de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Bioengenharia

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

Electrotaxis baseados em microfluídica para análise quantitativa On-demand de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Microscopia de folha de luz isotrópica e análise de linhagem de células automatizadas para catalogar Caenorhabditis elegans Embryogenesis com resolução subcelular

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Biologia do Desenvolvimento

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Um método de imagem semi-alta-fonte e um kit de ferramentas de visualização de dados para analisar o desenvolvimento embrionário de C. elegans

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Microchambers agarose para Imagem de cálcio a longo prazo de<em&gt; Caenorhabditis elegans</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Neurociência

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Biologia

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

Usando um Chip olfativo Microfluidic adaptado para a imagem da atividade Neuronal em resposta aos Pheromones em neurônios de cabeça masculino C. Elegans

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Utilizando um dispositivo microfluídica para estimulação mecânica e alta resolução de imagem de c. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Um chip microfluídico simples para crescimento a longo prazo e imagem de Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Cultura de Longo Prazo de Caenorhabditis elegans Individuais em Meio Sólido para Monitoramento Longitudinal de Fluorescência e Intervenções Aversivas

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Uma plataforma Microfluidic para Imaging Longitudinal em Caenorhabditis elegans

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