Este trabalho foi financiado pelo Australian Research Equipamentos Infra-estrutura do Conselho Linkage e Instalações Grant LEO668421 e Australian Research Council Linkage Projeto Grant LP0669471. Os autores agradecem a colaboração do Instituto de Tecnologia de Kanazawa e Elétrica Yokogawa Corporation em que estabelece o KIT-Macquarie Laboratório de Pesquisas sobre o Cérebro.

Essas orientações descrevem equipamentos e procedimentos para a medição da função cognitiva do cérebro em pré-escolares utilizando magnetoencefalografia (MEG). Em primeiro lugar, discutimos as diretrizes gerais e questões que devem ser considerados aquando da realização de testes experimentais da função cognitiva em crianças. Segundo, nós descrevemos um protocolo desenvolvido para alinhar os requisitos da MEG coleta de dados com as capacidades motivacionais e comportamentais de quatro anos de idade. 1. Considerações gerais para o estudo de crianças com MEG MEG pesquisadores enfrentam uma série de desafios únicos acordados quando se trabalha com crianças saudáveis. Em primeiro lugar, um ambiente de laboratório austero e funcional é provável para intimidar ou assustar crianças pequenas. Segundo, os requisitos básicos de um estudo experimental MEG incluem restrição de movimento e participar ativamente de uma tarefa por um período prolongado, as condições que muitos adultos encontrar tedioso e desconfortável. Ao estudar crianças torna-se imperativo para adaptar os procedimentos experimentais para as capacidades e limitações das crianças. Nesta seção, descrevemos os passos iniciais envolvidos na preparação do sujeito para participar de uma sessão de aquisição de dados. Sessão de familiarização 1,1 / Formação: Para pré-escolares de uma sessão de familiarização / formação está agendada antes da sessão real de aquisição de dados. Esta sessão permite que a criança se familiarizar com o ambiente e os pesquisadores em seu próprio ritmo. Introduzimos a criança eo pai para os pesquisadores e familiarizá-los com o ambiente de laboratório e rotinas. Ao final da sessão introdutória, o pai ea criança entender todas as etapas envolvidas em participar e entender o que eles devem esperar durante a gravação de dados. Se durante a primeira visita, uma criança aparece tímido ou ansioso, temos um período de tempo de reprodução até que eles se sentem mais confortáveis. Então, procedemos com a introdução de todas as etapas. 1,2 Explicando o meio ambiente: Para explicar o ambiente MEG para a criança, que empregam um tema que fornece uma justificação criança-amigável para o ambiente. Por exemplo, o sistema MEG é uma nave espacial à espera de levar as crianças em uma aventura espacial, onde alguns eventos estranhos podem ocorrer. O laboratório também foi decorada com adesivos de parede e brinquedos (Fig. 1), e tem uma sala de jogos dedicada. Ao falar com a criança, usamos a linguagem que as crianças podem facilmente compreender como o &quot;congelamento&quot; em vez de &quot;movimento minimizando &#39;, e&#39; capacete de astronauta&quot;, em vez de &#39;cap-coil marcador. 1,3 Explicando os procedimentos para a criança e aos pais: Nós mostramos a criança e os pais os passos que são tomadas antes que a criança vai para dentro da sala de blindagem magnética (MSR). Um dos primeiros passos é encaixar uma tampa marcador bobina e cinco na cabeça do sujeito; estas bobinas indicar a posição da cabeça do sujeito dentro do capacete MEG. Com a ajuda da criança, que digitalizar a forma da cabeça de um boneco, e então nós o convidamos a criança e os pais para acompanhar o boneco para o MSR / nave espacial. Uma vez dentro do MSR, colocamos a cabeça do boneco dentro do capacete para assistir a um filme. Em seguida, pedir que a criança se quiserem assistir o filme com o boneco. Se a criança está confortável e relaxado e seu pai está satisfeito com nossos protocolos, podemos convidá-los para jogar um jogo no interior da nave espacial na próxima vez que visitar o nosso laboratório. 1,4 Workbooks: Temos pastas de trabalho com base no tema aventura espacial que mostra as etapas crianças terão durante a sessão de testes. As crianças são dadas essas worksbooks para levar para casa e trabalhar através deles com seus pais em preparação para a próxima visita. O objetivo das pastas de trabalho é enfatizar a importância dos três requisitos principais para o teste: a) a prevenção de qualquer material ferroso no interior do MSR, b) a colocação de tampa da bobina marcador para garantir que os dados precisos co-registro e c) e os importância de se manter ainda durante a digitalização e aquisição de dados. 2. Um protocolo para medição de sinais de MEG desde a pré-escolares 2,1 Os equipamentos utilizados durante a aquisição de dados ea tarefa experimental Marcador de bobina-cap: a fim de alinhar os dados MEG com informações estruturais do sujeito (por exemplo, forma da cabeça de ressonância magnética ou digitalizados), um conjunto de cinco bobinas são colocadas em uma touca de natação que a criança usa dentro do capacete MEG. Estas bobinas funcionam como pontos de referência e as suas posições são comparados antes e depois da tarefa de medir o quanto a cabeça do sujeito se moveu durante a aquisição. Digitalizador: Antes de aquisição de dados, a posição de cinco bobinas marcador eo formato da cabeça do sujeito é digitalizado usando um F Polhemusastrak ® digitalizador (Colchester, VT). O digitador é composto por um digitalizador caneta, três receptores, um transmissor e uma unidade de controlo de movimentos. Este processo de medidas a localização das bobinas. Mais tarde, esses locais são comparados com as informações de localização obtida por MEG para alinhar os dados adquiridos magnético e formato da cabeça. Sistema de criança MEG: aquisição de dados é feita usando uma cabeça de todo o sistema de criança MEG (KIT, Kanazawa, Japão) [2]. Os dados são adquiridos continuamente a uma taxa de amostragem de 1000Hz, com um passa-banda de filtro online entre 0,03 Hz e 200Hz. Posições bobina marcador são obtidos antes e após as gravações para medir o movimento da cabeça. Os dados são rejeitados se o movimento da cabeça é maior do que 5mm. MEG sistema de periféricos: estímulos Experimental são controlados com um PC usando ® Presentation (Sistemas Neurocomportamentais, Albany, CA). Informações visuais são entregues por um projetor que fica fora da MSR e é refletida na tela MSR. Estímulos auditivos são entregues pelo Modelo de Pesquisa Etymotic ER-30 fones de ouvido equipados com fones de inserção do bebê do tamanho de espuma (Etymotic Research Inc., Elk Grove Village, IL). A criança é constantemente monitorado por câmera de circuito fechado e o experimentador se comunica com a criança através de um microfone. 2,2 aquisição de dados: as sessões de gravação de dados envolvem as seguintes etapas: Estágio I: Antes da chegada da criança É importante ter todos os computadores, software e equipamentos pronto para começar a experiência, logo que a criança chega. Como recomendação geral, dois investigadores experientes devem estar presentes enquanto a criança e os pais estão no laboratório. Comunicação clara entre os pais, pesquisador e criança são cruciais para o sucesso do experimento. Estágio II: Depois que a criança chega <ol><li> Formulários de consentimento são assinados e os experimentadores verificar se os pais têm dúvidas. </li><li> O experimentador verifica se a criança se lembra de todas as etapas introduzidas durante a primeira reunião ou nos folhetos e reintroduz as que não são lembrados. A importância do congelamento ou minimizar o movimento durante a aquisição de digitalização e de dados é enfatizada. Isto pode ser demonstrado através de um fantoche. </li><li> Então, o experimentador verifica se a criança é portador de qualquer material ferroso (roupas com manchas glitter, jóias, brinquedos esquecidos dentro de bolsos, alfinetes de cabelo, etc.) Se eles carregam uma peça de roupa que contém material ferroso, nós fornecê-los com roupas MEG segura. Se o pai quiser acompanhar a criança durante o experimento, podemos garantir que eles também retirar seu material ferroso. </li></ol> Estágio III: Getting a criança pronto para executar a tarefa dentro do MSR <ol><li> O experimentador se encaixa a tampa que prende os 5 bobinas marcador. </li><li> O experimentador digitaliza a localização das bobinas marcador e forma da criança cabeça. A fim de manter as cabeças em uma posição fixa, usamos um colar cervical, que foi equipado com três receptores. É importante reforçar a idéia de minimizar o movimento ou &quot;congelamento&quot; regularmente para obter uma forma boa cabeça. A criança pode ser recompensado com um adesivo para fazer um bom trabalho no &quot;congelamento&quot;. </li><li> A criança é convidada a entrar no MSR. Fones de ouvido estão inseridos e que a criança é posicionada no capacete dewar MEG em posição supina. </li><li> Quando a criança está deitada confortavelmente com a cabeça dentro do capacete MEG, as bobinas marcador estão ligados à sua unidade de potência ea porta MSR está fechado. Um experimentador permanece (e também um pai, se desejar) no quarto durante toda a sessão. </li><li> Para o entretenimento durante a instalação e entre os blocos de coleta de dados, um filme ou desenho animado pode ser projetada na tela MSR retro-projeção. </li></ol> Estágio IV: A execução do experimento <ol><li> Assim que a porta está fechada MSR, SQUIDs estão engajados e aquisição de dados começa. </li><li> Como a criança assiste a um filme, um coregistration pré-teste é realizado para se certificar de que os sinais de MEG pode ser co-registrados para o formato da cabeça digitalizados. Este pré-teste consiste de combinar a posição das bobinas marcador obtidos durante a digitalização com a posição indicada pelo sistema de MEG. </li><li> Uma vez que a mostra primeiro teste que a cabeça da criança está corretamente posicionado dentro do capacete, o pesquisador pode iniciar o experimento: (a) o filme é mudo; (b) o pesquisador pergunta ao filho se eles estão prontos para &quot;jogar o jogo&quot;; (c) uma vez que o jogo começa, o experimentador fornece regularmente a criança com feedback. Por exemplo, uma criança pode ser convidado a ouvir uma série de frases, e para repetir uma palavra-alvo, tais como &#39;queijo&#39;, assim que ouvi-lo. Estas palavras-alvo são incorporados em um subconjunto de captura trials que sonda continuou a atenção para a tarefa, mas não são incluídos na análise final. </li><li> Uma vez que a tarefa é concluída, outra medida da bobina marcador é realizado para monitorar a posição da cabeça e do movimento. </li><li> Uma vez que o SQUIDs são desligados, a porta é aberta e MSR a criança é convidada a uma sala de jogos onde se pode escolher um prêmio para o seu bom trabalho. </li></ol> 3. Resultados representativos Um estudo representativo em nosso laboratório usado este protocolo para estudar o desenvolvimento da lógica na linguagem infantil. Vinte e seis crianças de 3-4 anos (idade média de 4 anos e 5 meses) participou em pelo menos uma sessão de aquisição. Dezessete dessas crianças voltaram para uma sessão de testes de segunda e cinco vieram para uma sessão de testes de terceiros. O movimento da cabeça média foi de menos de 3,7 mm (sd 3,1 mm), bem dentro do limite de 5 mm. Movimentos maiores que 5mm ocorreu em 4,5% das sessões, com variação variando de 5,5 mm a 15 mm (média = 8,6 milímetros, dp = 2,5 mm). <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="Figura 1" /> Figura 1. O capacete MSR e MEG foram decorados com adesivos e brinquedos de pelúcia. <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="Figura 2" /> Figura 2. Quatro menina anos de idade, em tamanho adulto MEG (esquerda). Observe o preenchimento no topo da cabeça para minimizar o movimento e da distância entre a cabeça eo capacete. Imagem da direita mostra menina mesmo no MEG criança.

<ol>
	<li>H&#228;m&#228;l&#228;inen, M., Hari, R., Ilmoniemi, R. J., Knuutila, J., Lounasmaa, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalography-+theory,+instrumentation,+and+applications+to+noninvasive+studies+of+the+working+human+brain.">Magnetoencephalography- theory, instrumentation, and applications to noninvasive studies of the working human brain.</a> Rev. Mod. Physics. 65 (2), 413-497 (1993).</li><li>Johnson, B. W., Crain, S., Thornton, R., Tesan, G., Reid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+brain+function+in+pre-school+children+using+a+custom+sized+whole-head+MEG+sensor+array.">Measurement of brain function in pre-school children using a custom sized whole-head MEG sensor array.</a> Clin. Neurophysiol. , (2009).</li><li>Kado, H., Higuchi, M., Shimogawara, M., Haruta, Y., Adachi, Y., Kawai, J., Ogata, H., Uehara, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetoencephalogram+system+developed+at+KIT.">Magnetoencephalogram system developed at KIT.</a> IEEE Trans. Appl. Supercond. 9, 4057-4062 (1999).</li><li>Uehara, G., Adachi, Y., Kawai, J., Shimogawara, M., Higuchi, M., Haruta, Y., Ogata, H., Kado, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Channel+SQUID+Systems+for+Biomagnetic+Measurement.">Multi-Channel SQUID Systems for Biomagnetic Measurement.</a> IEICE Trans. Electr. E86-C, 43-54 (2003).</li></ol>

Este protocolo é projetado para facilitar as medidas de função cognitiva do cérebro em indivíduos saudáveis ​​e desperta 4 anos de idade. Usamos um sistema de tamanho personalizado MEG projetado para se encaixar as cabeças dos pré-escolares. Com este sistema, uma criança-friendly ambiente de laboratório, e um protocolo adaptado às capacidades e motivações das crianças, somos capazes de medir a função cognitiva do cérebro em uma faixa etária para a qual não é actualmente muito poucos dados. O sistema de KIT-Macquarie é o costume primeiro sistema todo cabeça de tamanho para uso com pré-escolares. Existem três principais vantagens do capacete personalizado dewar para estudar as crianças. Primeiro, o menor raio de configuração do sensor traz os sensores SQUID na faixa dos sinais neuromagnetic de cabeças de crianças s. Segundo, a menor capacete permite a inserção plena da cabeça de uma criança no dewar. Plena inserção é impedida em capacetes adulto dewar por causa da menor distância coroa a ombro em crianças (ver fig. 2). Estes dois fatores são fundamentais na gravação de atividade cerebral usando MEG porque os sinais neuromagnetic atenuar rapidamente com a distância. Terceiro, a aids infantil personalizado capacete no posicionamento simétrico da cabeça e limita a liberdade de movimento da cabeça da criança dentro do dewar. Estas características significativamente facilitar a configuração, posicionamento e medição de sinais de MEG das crianças. O advento de tamanho personalizado criança MEG sistema é um avanço importante para a neurociência cognitiva. Alternativas técnicas de neuroimagem funcional radiofármacos que empregam ou fortes campos magnéticos pode não ser adequado para uso de rotina com as crianças. Desde MEG é uma técnica de medição inteiramente passivo não há risco concebível para o desenvolvimento de tecidos. Há actualmente muito poucos dados MEG de pré-escolares saudáveis, porque os sistemas convencionais MEG adultos não são bem adaptados para utilizar com as crianças. Sistemas criança MEG nos permitirá estudar a função cerebral em idades em que uma estruturação dramática da atividade do cérebro está ocorrendo, oferecendo novas percepções sobre o desenvolvimento normal e patológico da função cognitiva.

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<td>Whole head child MEG</td>
<td>&nbsp;</td>
<td>Kanazawa Institute of Technology (Kanazawa) and Yokogawa Electric Corporation (Tokyo)</td>
<td>Model PQ1064R-N2m</td>
<td>&nbsp;</td>
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<td>Magnetically shielded room</td>
<td>&nbsp;</td>
<td>Fujihara Co. Ltd., (Tokyo)</td>
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<td>Digitiser</td>
<td>&nbsp;</td>
<td>Polhemus (Colchester, VT)</td>
<td>Fastrack</td>
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<td>&nbsp;</td>
<td>Neurobehavioral Systems (Albany, CA)</td>
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<td>Earphones</td>
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<td>Etymotic Research Inc. (Elk Grove Village, IL)</td>
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measurement of neuromagnetic brain function in pre-school children with custom sized meg

Magnetoencefalografia é uma técnica que detecta campos magnéticos associados com a atividade cortical [1]. A atividade eletrofisiológica do cérebro gera campos elétricos - que podem ser gravados usando eletroencefalografia (EEG) - e seus concomitantes campos magnéticos - detectada por MEG. MEG sinais são detectados por sensores especializados conhecidos como dispositivos supercondutores de interferência quântica (SQUID). Sensores supercondutores requerem refrigeração com hélio líquido a -270 ° C. Eles estão contidos dentro de um capacete vacumm isolado chamado de dewar, que é preenchido com líquido. LULAS são colocados em posições fixas no interior do capacete dewar no refrigerante de hélio, e cabeça de um sujeito é colocado dentro do capacete dewar para medições de MEG. O capacete dewar devem ser dimensionados para satisfazer as restrições opostas. Claramente, ele deve ser grande o suficiente para caber a maioria ou todos os cabeças na população que será estudada. No entanto, o capacete também deve ser pequeno o suficiente para manter a maioria dos sensores SQUID dentro do alcance dos campos minúsculos cerebral que eles são a medida. Sistemas convencionais de todo-cabeça MEG são projetados para acomodar mais de 90% dos chefes de adultos. Sistemas No entanto adultos não são adequados para medir a função cerebral em idade pré-escolar chidren cujas cabeças têm um raio de vários centímetros menores do que os adultos. O KIT-Cérebro Macquarie Research Laboratory na Universidade Macquarie usa um sistema personalizado MEG tamanho para caber na cabeça de crianças pré-escolares. Este sistema tem 64 crianças de primeira ordem gradiómetros axial com uma base de 50 mm [2] e está contido dentro de uma sala magneticamente blindado (MSR), juntamente com um sistema convencional MEG tamanho adulto [3,4]. Existem três principais vantagens do capacete personalizado dewar para estudar as crianças. Primeiro, o menor raio de configuração do sensor traz os sensores SQUID na faixa dos sinais neuromagnetic de cabeças das crianças. Segundo, a menor capacete permite a inserção plena da cabeça de uma criança no dewar. Plena inserção é impedida em capacetes adulto dewar por causa da menor distância coroa a ombro nas crianças. Estes dois fatores são fundamentais na gravação de atividade cerebral usando MEG porque os sinais neuromagnetic atenuar rapidamente com a distância. Terceiro, a aids infantil personalizado capacete no posicionamento simétrico da cabeça e limita a liberdade de movimento da cabeça da criança dentro do dewar. Quando usado com um protocolo que alinha os requisitos de coleta de dados com as capacidades motivacionais e comportamentais das crianças, esses recursos de forma significativa facilitar a configuração, posicionamento e medição de sinais de MEG.

Watch this Scientific Journal Video about Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG at JoVE.com

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

O advento dos sistemas MEG tamanho para crianças abre novas e importantes oportunidades para estudar o desenvolvimento do cérebro. O novo sistema, juntamente com um protocolo que alinha os requisitos experimental com as capacidades das crianças, pode ser usado para estudar processos cognitivos e de linguagem em crianças saudáveis, com idades entre acordado 05:57.

Medição da função cerebral Neuromagnetic Em pré-escolares Com Personalizado MEG Sized

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

measurement-neuromagnetic-brain-function-pre-school-children-with

Nanyang Technological University

Research

JoVE Journal

Biology

16.8K visualizações.  Macquarie University. O advento dos sistemas MEG tamanho para crianças abre novas e importantes oportunidades para estudar o desenvolvimento do cérebro. O novo sistema, juntamente com um protocolo que alinha os requisitos experimental com as capacidades das crianças, pode ser usado para estudar processos cognitivos e de linguagem em crianças saudáveis, com idades entre acordado 05:57.

Vídeo: Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Longo prazo células de mamíferos utilizando imagens do Largo-Campo Microscopia

Biologia

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

Medição in vivo Micro-circulação no músculo esquelético por Intra-vital Microscopia

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Explorando Funções Cognitivas em Bebés, Crianças e Adultos com Espectroscopia no Infravermelho Próximo

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Performing Custom MicroRNA Microarray Experiments

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally 2, 3. miRNAs control a broad range of biological processes 1. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis 4, 5. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.
Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

A realização de experimentos de Microarray personalizado MicroRNA

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

Genoma Edição com CompoZr personalizados Nucleases dedo de zinco (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Medição de Quimiotaxia celulares com ECIS / Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Medição de força durante a contração para avaliar a função muscular em larvas do peixe

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

A medição da função do músculo liso nas Isolado tecido de banho-aplicativos para Farmacologia Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstituição funcional e atividade do canal Medidas de purificada tipo selvagem e mutante CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Preparação da amostra e a imagem latente de Exosomes por microscopia eletrônica de transmissão

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...