Este trabajo fue apoyado por el australiano de investigación de equipos de infraestructura del Consejo de Vinculación y dar facilidades LEO668421 Consejo Australiano de Investigación y Vinculación de proyectos de subvención LP0669471. Los autores agradecen la colaboración del Instituto de Tecnología de Kanazawa y Yokogawa Electric Corporation en el establecimiento del Laboratorio de Cerebro KIT-Macquarie Research.

Estas directrices describen los equipos y procedimientos para medir la función cerebral cognitiva en niños en edad preescolar mediante magnetoencefalografía (MEG). En primer lugar, sobre las orientaciones generales y las cuestiones que deben ser considerados en el ejercicio de las pruebas experimentales de la función cognitiva en los niños. En segundo lugar, se describe un protocolo desarrollado para alinear los requisitos de la MEG recopilación de datos con la capacidad de motivación y de comportamiento de cuatro años de edad. 1. Consideraciones generales para el estudio de los niños pequeños con MEG MEG investigadores se enfrentan a una serie de retos únicos cuando se trabaja con niños sanos despiertos. En primer lugar, un entorno de laboratorio cruda y funcional es probable que para intimidar o asustar a los niños pequeños. En segundo lugar, los requisitos básicos de un estudio experimental MEG incluyen la restricción de movimiento y de forma activa asistiendo a una tarea durante un período prolongado, las condiciones que muchos adultos encuentran tedioso e incómodo. Al estudiar a los niños, se vuelve imperativo adecuar los procedimientos experimentales de las capacidades y limitaciones de los niños. En esta sección se describen los primeros pasos involucrados en la preparación de la materia a participar en una sesión de adquisición de datos. 1.1 Familiarización / Sesión de Entrenamiento: Para niños en edad preescolar una sesión de familiarización / formación está prevista antes de la sesión de adquisición de datos reales. Esta sesión permite que el niño se familiarice con el medio ambiente y los investigadores a su propio ritmo. Nos introducimos en el niño y los padres de los investigadores se familiaricen con el entorno de laboratorio y las rutinas. Al final de la sesión de introducción a los padres y el niño a entender todos los pasos involucrados en participar y entender lo que les espera durante la grabación de datos. Si durante la primera visita, el niño parece tímido o ansioso, tenemos un periodo de tiempo de juego hasta que se sientan más cómodos. A continuación, se procede a la introducción de todos los pasos. 1.2 Explicar el medio ambiente: Para explicar el medio ambiente MEG para el niño, que emplean a un tema que proporciona una justificación favorable a los niños de los alrededores. Por ejemplo, el sistema de MEG es una nave espacial a la espera de llevar a los niños en una aventura espacial, donde algunos eventos desconocidos pueden ocurrir. El laboratorio también ha sido decorado con pegatinas y juguetes (Fig. 1), y se ha dedicado una sala de juegos. Al hablar con el niño, el uso del lenguaje que los niños pueden entender fácilmente como el &quot;congelamiento&quot; en lugar de &quot;minimizar el movimiento, y el casco de astronauta&quot; en lugar de &quot;marcador de bobina cap &#39;. 1.3 Explicar los procedimientos para el niño y los padres: Se muestra el niño y los padres las medidas que se toman antes de que el niño va dentro de la sala con blindaje magnético (MSR). Uno de los primeros pasos es instalar un marcador de cinco-coil límite a la cabeza del sujeto, estas bobinas indican la posición de la cabeza del sujeto dentro del casco MEG. Con la ayuda del niño, que digitalizar la forma de la cabeza de una marioneta, y luego invitar a los niños y los padres para acompañar a la marioneta a la MSR / nave espacial. Una vez dentro del MSR, ponemos la cabeza del títere en el interior del casco para ver una película. A continuación, preguntar al niño si quiere ver la película con el títere. Si el niño se sienta cómodo y relajado, y su padre está satisfecho con nuestros protocolos, los invitamos a jugar a un juego interior de la nave la próxima vez que visite nuestro laboratorio. 1.4 libros: Tenemos libros basados ​​en el tema aventura espacial que muestra los pasos los niños se llevará a durante la sesión de pruebas. Los niños reciben estos worksbooks para llevar a casa y trabajar a través de ellos con sus padres en la preparación de la próxima visita. El objetivo de los cuadernos de trabajo es poner de relieve la importancia de los tres requisitos principales para la prueba: a) la evitación de cualquier material ferroso en el interior del MSR, b) la colocación de la tapa marcador de bobina para conseguir datos precisos del co-registro y c) y el importancia de permanecer quieto durante la digitalización y la adquisición de datos. 2. Un protocolo para la medición de señales de MEG desde el nivel preescolar los niños de 2.1 El equipo utilizado en la adquisición de datos y la tarea experimental Marcador de la bobina de la PAC: el fin de alinear los datos MEG con la información estructural del sujeto (por ejemplo, forma magnética de la cabeza o digitalizados), un conjunto de cinco bobinas se colocan en una gorra de natación que el niño lleva dentro del casco MEG. Estas bobinas actúan como puntos de referencia y sus posiciones se comparan antes y después de la tarea de medir la cantidad de la cabeza del sujeto se ha movido durante la adquisición. Digitalizador: Antes de la adquisición de datos, la posición de cinco bobinas de marcador y la forma de la cabeza del sujeto se digitaliza usando un F Polhemusastrak ® digitalizador (Burlington, VT). El digitalizador consta de un digitalizador de pluma, tres receptores, un transmisor y una unidad de seguimiento de movimiento. Este proceso mide la ubicación de las bobinas. Más tarde, estos lugares se comparan con la información de ubicación obtenida por MEG para alinear los datos adquiridos magnético y forma de la cabeza. Niño sistema de MEG: La adquisición de datos se realiza mediante una cabeza de todo el sistema de MEG niño (KIT, Kanazawa, Japón) [2]. De datos se realiza continuamente a una velocidad de muestreo de 1000 Hz, con una línea de paso de banda de filtro entre 0,03 Hz y 200 Hz. Posiciones de marcador de la bobina se obtienen antes y después de las grabaciones para medir el movimiento de cabeza. Los datos son rechazados si el movimiento de la cabeza es mayor de 5 mm. MEG sistema de periféricos: los estímulos experimentales se controlan con una PC usando Presentación ® (Sistemas neuroconductuales, Albany, CA). Entradas visuales son entregados por un proyector que se encuentra fuera del MSR y se refleja en la pantalla del MSR. Los estímulos auditivos son entregados por el Modelo de Investigación Etymotic ER-30 equipado con auriculares insertar auriculares bebé del tamaño de la espuma (Etymotic Research Inc., Elk Grove Village, IL). El niño es constantemente monitoreado por cámaras de circuito cerrado y el experimentador se comunica con el niño a través de un micrófono. 2.2 Adquisición de datos: Las sesiones de grabación de datos implica las siguientes etapas: Etapa I: Antes de la llegada del niño Es importante que todos los equipos, software y equipos listos para comenzar el experimento, tan pronto como el niño llega. Como recomendación general, dos investigadores con experiencia debe estar presente mientras el niño y sus padres están en el laboratorio. Una comunicación clara entre el investigador, padre e hijo son cruciales para el éxito del experimento. Fase II: Después de que el niño llega <ol><li> Los formularios de consentimiento firmados y los experimentadores comprobar si los padres tienen preguntas. </li><li> El experimentador comprueba si el niño recuerda todos los pasos introducido durante la primera reunión o en los folletos y reintroduce las que no se recuerdan. La importancia de la congelación o minimizar el movimiento durante la adquisición de la digitalización y los datos se pone de relieve. Esto se puede demostrar con un títere. </li><li> Entonces, el investigador comprueba si el niño es portador de material ferroso (la ropa con manchas brillantes, joyas, juguetes olvidados dentro de los bolsillos, horquillas para el cabello, etc.) Si llevan un pedazo de ropa que contiene material ferroso, les damos ropa MEG seguro. Si el padre desea para acompañar al niño durante el experimento, nos aseguramos de que también eliminan los materiales ferrosos. </li></ol> Etapa III: Cómo el niño listo para realizar la tarea en el interior del MSR <ol><li> El experimentador se ajusta a la tapa que contiene el 5 bobinas marcador. </li><li> El experimentador digitaliza la ubicación de las bobinas del marcador y la forma de la cabeza del niño. A fin de mantener la cabeza en una posición fija, se utiliza un cuello ortopédico que ha sido equipado con tres receptores. Es importante reforzar la idea de reducir al mínimo el movimiento o la &quot;congelación&quot; con regularidad para obtener una buena forma de la cabeza. El niño puede ser recompensado con una pegatina para hacer un buen trabajo en el &quot;congelamiento&quot;. </li><li> El niño es invitado a entrar en el MSR. Auriculares se insertan y que el niño se coloca en el casco MEG termo en posición supina. </li><li> Una vez que el niño está acostado cómodamente con la cabeza dentro del casco MEG, las bobinas de marcador está conectado a su unidad de poder y la puerta se cierra MSR. Un experimentador sigue siendo (y también uno de los padres, si se desea) en la sala durante toda la sesión. </li><li> Para el entretenimiento durante la instalación y entre los bloques de recopilación de datos, una película de dibujos animados o pueden ser proyectadas en el MSR de retroproyección de la pantalla. </li></ol> Etapa IV: Ejecución del experimento <ol><li> Una vez que la puerta está cerrada MSR, calamares se dedican y se inicia la adquisición de datos. </li><li> A medida que el niño ve una película, un corregistro pre-test se lleva a cabo para asegurarse de que las señales de MEG puede ser corregistradas a la forma de la cabeza digitalizados. Este pre-test consiste en el casamiento de la posición de las bobinas marcador obtenido durante la digitalización con la posición mostrada por el sistema de MEG. </li><li> Una vez que la muestra la primera prueba de que la cabeza del niño esté correctamente colocado en el interior del casco, el investigador puede iniciar el experimento: (a) de la película está en silencio, (b) el investigador pregunta al niño si no están dispuestos a &quot;jugar el juego&quot;; (c) una vez que se inicia el juego, el experimentador proporciona regularmente al niño con retroalimentación. Por ejemplo, un niño se le puede pedir a escuchar una serie de oraciones, y repetir una palabra objetivo como &quot;queso&quot;, tan pronto como lo oyen. Estas palabras de destino están integrados en un subconjunto de la captura de tresals que la sonda continuó su atención a la tarea, pero no están incluidos en el análisis final. </li><li> Una vez finalizada la tarea, otra medida de la bobina marcador se lleva a cabo para controlar la posición de la cabeza y el movimiento. </li><li> Una vez que los calamares no están conectados, la puerta del MSR se abre y el niño es invitado a una sala de juegos donde pueden elegir un premio por su buen trabajo. </li></ol> 3. Los resultados representativos Un estudio representativo en nuestro laboratorio utiliza este protocolo para estudiar el desarrollo de la lógica en el lenguaje infantil. Veintiséis niños de 3-4 años (media de edad de 4 años y 5 meses) participaron en la sesión de adquisición de al menos uno. Diecisiete de esos niños regresó para una segunda sesión de pruebas y cinco vino para una sesión de pruebas de terceros. Movimiento de la cabeza promedio fue de menos de 3,7 mm (DE 3,1 mm), y en el umbral de 5 mm. Movimientos de más de 5 mm se produjo en el 4,5% de las sesiones, con una variación que van desde 5,5 mm a 15 mm (media = 8.6 mm, sd = 2,5 mm). <img src="/files/ftp_upload/1693/1693fig1.jpg" alt="Figura 1" /> Figura 1. El casco MSR y MEG han sido decoradas con pegatinas y juguetes de peluche. <img src="/files/ftp_upload/1693/1693fig2.jpg" alt="Figura 2" /> Figura 2. Cuatro años de edad, niña de tamaño adulto MEG (izquierda). Tenga en cuenta el relleno en la parte superior de la cabeza para minimizar el movimiento y la distancia entre la cabeza y el casco. Imagen de la derecha muestra la misma chica en el MEG niño.

<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 está diseñado para facilitar las mediciones de la función cerebral cognitiva en personas sanas y despierto de 4 años de edad. Utilizamos un sistema de tamaño personalizado MEG diseñado para adaptarse a los jefes de pre-escolares. Con este sistema, un niño amigable entorno de laboratorio, y un protocolo adaptado a las capacidades y motivaciones de los niños, que son capaces de medir la función cerebral cognitiva en un rango de edad para la que existe en la actualidad muy pocos datos. El sistema KIT-Macquarie es la primera de todo el sistema de la cabeza a medida de tamaño para su uso con pre-escolares. Hay tres ventajas principales del casco personalizado termo para el estudio de los niños. En primer lugar, el radio más pequeño de la configuración del sensor lleva los sensores SQUID dentro del rango de las señales de neuromagnetic de cabeza de los niños s. En segundo lugar, el casco más pequeño permite la inserción completa de la cabeza de un niño en el termo. Inserción completa es prevenir en adultos cascos dewar debido a la corona más pequeña para asumir la distancia en los niños (ver fig. 2). Estos dos factores son fundamentales en el registro de actividad cerebral mediante MEG porque las señales neuromagnetic atenúan rápidamente con la distancia. En tercer lugar, las ayudas niño personalizado casco en la posición simétrica de la cabeza y los límites de la libertad de movimiento de la cabeza del niño dentro del dewar. Estas características facilitan significativamente la configuración, posicionamiento y medición de señales de MEG de los niños. El advenimiento de la costumbre de tamaño infantil MEG sistema es un avance importante para la neurociencia cognitiva. Alternativas técnicas de neuroimagen funcional que emplean radiofármacos o fuertes campos magnéticos pueden no ser apropiados para el uso habitual con niños pequeños. Desde el MEG es una técnica de medición totalmente pasiva no hay ningún riesgo concebible para el desarrollo de los tejidos. En la actualidad existe muy poca información MEG de niños sanos en edad preescolar, ya que los sistemas convencionales para adultos MEG no están bien adaptados para utilizar con los niños. Niño sistemas MEG nos permitirá estudiar la función cerebral en edades donde una estructuración dramática de la actividad cerebral se produce, proporcionando nuevos conocimientos sobre el desarrollo normal y patológico de la función 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>
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<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>Neurobehavioral Systems (Albany, CA)</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

La magnetoencefalografía es una técnica que detecta los campos magnéticos asociados con la actividad cortical [1]. La actividad electrofisiológica del cerebro genera campos eléctricos - que se puede grabar utilizando la electroencefalografía (EEG) - y sus campos magnéticos concomitante - detectado por MEG. MEG señales son detectadas por sensores especializados conocidos como dispositivos superconductores de interferencia cuántica (SQUID). Sensores superconductores requieren enfriamiento con helio líquido a -270 ° C. Están contenidas dentro de un casco estela aislado llamado Dewar, que está lleno de líquido. CALAMARES se colocan en posiciones fijas en el interior del casco termo en el refrigerante de helio, y la cabeza de un sujeto se encuentra dentro del casco termo para las mediciones de MEG. El casco Dewar debe ser diseñado para satisfacer las limitaciones de oposición. Está claro que debe ser lo suficientemente grande como para caber la mayoría o la totalidad de los jefes de la población que se estudiará. Sin embargo, el casco debe ser lo suficientemente pequeño como para mantener la mayoría de los sensores SQUID dentro del alcance de los pequeños campos cerebral que se van a medir. Convencionales de todo el cabeza de los sistemas de MEG se han diseñado para dar cabida a más del 90% de los jefes de adultos. Sin embargo, los sistemas de adultos no son adecuados para la medición de la función cerebral en la enseñanza preescolar Chidren cuyas cabezas tienen un radio de varios centímetros más pequeño que los adultos. El KIT-Macquarie cerebro Laboratorio de Investigación de la Universidad de Macquarie utiliza un sistema personalizado MEG tamaño para adaptarse a las cabezas de los niños en edad preescolar. Este sistema cuenta con 64 niños de primer orden gradiómetros axial con una línea de base de 50 mm [2] y está contenida dentro de un ambiente protegido magnéticamente (MSR), junto con un adulto de tamaño convencional sistema de MEG [3,4]. Hay tres ventajas principales del casco personalizado termo para el estudio de los niños. En primer lugar, el radio más pequeño de la configuración del sensor lleva los sensores SQUID dentro del rango de las señales de neuromagnetic de cabeza de los niños. En segundo lugar, el casco más pequeño permite la inserción completa de la cabeza de un niño en el termo. Inserción completa es prevenir en adultos cascos dewar debido a la corona más pequeña para asumir la distancia en los niños. Estos dos factores son fundamentales en el registro de actividad cerebral mediante MEG porque las señales neuromagnetic atenúan rápidamente con la distancia. En tercer lugar, las ayudas niño personalizado casco en la posición simétrica de la cabeza y los límites de la libertad de movimiento de la cabeza del niño dentro del dewar. Cuando se utiliza con un protocolo que se alinea con los requisitos de recopilación de datos con la capacidad de motivación y comportamiento de los niños, estas características de manera significativa facilitar la instalación, colocación y medición de señales 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

El advenimiento de los sistemas de MEG de tamaño para los niños pequeños abre nuevas e importantes oportunidades para estudiar el desarrollo del cerebro. El nuevo sistema, junto con un protocolo experimental que se alinea con los requisitos de las capacidades de los niños, puede ser usado para estudiar los procesos cognitivos y del lenguaje en los niños sanos, despiertos tres a seis años.

La medición de la función cerebral neuromagnetic En Pre-escolar Niños con tamaño personalizado MEG

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

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16.8K vistas.  Macquarie University. El advenimiento de los sistemas de MEG de tamaño para los niños pequeños abre nuevas e importantes oportunidades para estudiar el desarrollo del cerebro. El nuevo sistema, junto con un protocolo experimental que se alinea con los requisitos de las capacidades de los niños, puede ser usado para estudiar los procesos cognitivos y del lenguaje en los niños sanos, despiertos tres a seis años.

Video: 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.

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

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.

En vivo la micro-circulación de medición en el músculo esquelético de Intra-vital Microscopía

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.

Explorar las funciones cognitivas en bebés, niños y adultos con Espectroscopia de Infrarrojo Cercano

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.

Realización de experimentos de microarrays personalizados microARN

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 Edición con CompoZr nucleasas de dedos de zinc personalizados (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.

La medición de la quimiotaxis celular con 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.

Medición de fuerza durante la contracción para evaluar la función muscular en larvas de pez cebra

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.

La medición de la función del músculo liso en el tejido aislado Bath-aplicaciones en Farmacología Investigación

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.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

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.

Preparación de la muestra y proyección de imagen de exosomas por microscopía electrónica de transmisión

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