Este trabajo fue apoyado por subvenciones del NIH DA018109 y MH78823, y el NIH Blueprint de Neurociencias básicas P30NS057105 subvención a la Universidad de Washington.

Cultura preparación <ol><li> Preparar cultivos disociados celular de las células del hipocampo de rata o ratón desde el primer día postnatal 0-3 animales 1. Las neuronas se adhieran a una monocapa de astrocitos subyacentes, que a su vez se adhiere a una capa de colágeno extiende sobre un cubreobjetos número de espesor 0. Placa de las neuronas a una densidad de aproximadamente 500 células por centímetro cuadrado. </li><li> Permita que las culturas de crecer durante 10-14 días para permitir el desarrollo y la maduración sináptica. El tratamiento de las culturas en el día 4 in vitro con la AraC antimitóticos para detener el crecimiento más gliales. Darles de comer en el día 5 in vitro con un intercambio de soportes de medio a medio con Neurobasal más suplemento B27 para mejorar la supervivencia neuronal. </li><li> Durante el período de cultivo, introducción de los tratamientos diseñados para alterar el número de sinapsis funcionales silenciados. Nos encontramos con que una forma cómoda para silenciar a un gran porcentaje (80%) de las sinapsis de glutamato es un tratamiento de 4 horas con 30 mM de potasio. Ya incubaciones con débiles estímulos despolarizantes inducen 2 silenciar similar. La estimulación de 4 horas con 50 M forskolin, un activador de la adenilato ciclasa, se puede utilizar para despertar a la población de los terminales de silencio al inicio del estudio 3. </li></ol> Visualización de sinapsis silenciosas <ol><li> Las células deben tener una densidad relativamente baja con los procesos de neuríticas bien separados en el día in vitro 10-14 con el fin de facilitar la visualización de los terminales sinápticos discretos mediante microscopía de fluorescencia. </li><li> Prepare una solución madre de 5 mM FM1-43FX en agua destilada. Las acciones deben ser mantenidos en la oscuridad a 4 ° C. Todos los experimentos con FM1-43FX también se debe realizar en la oscuridad. </li><li> Desafío de las culturas durante 2 minutos con 45 mM de cloruro de potasio en una solución salina tamponada con HEPES que contenía 100 mM de cloruro de sodio, cloruro de calcio 2 mM, 1 mM de cloruro de magnesio, 10 mM de glucosa, y 10 M FM1-43FX. Esta solución también debe contener una M y 25 M NBQX D-APV para bloquear los receptores postsinápticos y prevenir la señalización recurrente. Este reto se marca terminales sinápticos que son capaces de exocitosis y endocitosis. El tiempo de desafío y resistencia están diseñados para causar al menos una ronda de la exocitosis de vesículas todos los disponibles en el grupo 4 de reciclaje. Sin embargo, el desafío no es suficiente para provocar el silenciamiento adicional de terminales. </li><li> Lave la cultura de 3 a 5 segundos solamente, utilizando la misma solución salina tamponada HEPES sin cloruro de potasio, pero con 500 M Advasep-7 para quitar el tinte no específicos 5. Tenga en cuenta que la sincronización de este paso es fundamental, ya la aplicación prolongada de Advasep-7 se traducirá en la pérdida de todas las etiquetas FM1-43FX. Siguiente lavado en solución salina tamponada HEPES sin Advasep-7 durante cinco intervalos de 2 minutos. </li><li> Fijar las células con paraformaldehído al 4% y el 0,2% de glutaraldehído en PBS, pH 7,4 durante 10 minutos. Lave las células una vez brevemente con PBS, y luego se incuban durante 15 minutos en solución de bloqueo que contienen 4% de suero normal de cabra y el 0,04% de Triton X-100 en PBS. Tenga en cuenta que FM1-43FX es también muy sensible a la cantidad de Triton X-100 se utiliza aquí y en los pasos posteriores. En términos generales, FM1-43FX etiquetado es el mejor en la ausencia de Triton X-100, pero un poco de detergente es necesaria para permeabilizar las células de tinción posterior. Se ha determinado empíricamente que el 0,04% Triton X-100 es óptimo para el examen de las sinapsis presináptico en silencio. </li><li> Diluir el principal vGluT-1 anticuerpo en solución de bloqueo a una concentración de 1:2500. Se incuban las células con agitación suave y / o rotación en los gases de vGluT-1 anticuerpo durante 3 horas a temperatura ambiente. Asegúrese de cubrir sus placas de cultivo con papel aluminio para evitar la decoloración del colorante FM1-43FX. </li><li> Lavar las células dos veces en PBS y se incuban las células con un anticuerpo anti-indias de cerdo conjugado con un fluoróforo con diferentes características espectrales de FM1-43FX para distinguir las dos manchas. Para nuestro experimento, vamos a utilizar Alexa 555-conjugado de anticuerpos anti-indias de cerdo a 1:500 en solución de bloqueo. Se incuban las células con el anticuerpo secundario mientras se agita durante 30 minutos a temperatura ambiente. </li><li> Lavar las células cuatro veces más en PBS y luego montar el cubreobjetos sobre un portaobjetos con una pequeña gota de Fluoromount-G. Asegúrese de orientar el cubreobjetos para que las células frente a la diapositiva. Deje que el que se sequen durante la noche. </li><li> Ahora estamos listos para tomar imágenes de las sinapsis activas e inactivas. Prepare un objetivo de aceite 60 x con una abertura de 1,4 numérica en un microscopio láser confocal de barrido. Coloque una diapositiva en el escenario, asegurando que el lado del cubreobjetos se enfrenta el objetivo. Después de enfocar la diapositiva, encontrará un campo de neuritas que no es demasiado denso. También se recomienda para evitar la imagen de cerca del soma celular, como residual FM1-43FX medio de contraste puede permanecer allí aún después de un prolongado lavado. Para nuestras imágenes experimentales, por lo general acercar a un tamaño de campo de 51 51 micras. </li><li> Adquirirun z-stack en un determinado campo que abarca una profundidad de 7,8 micrones, con 27 0,3 micrones pasos. Este rango es suficiente para cubrir el espesor de las neuritas en nuestra cultura. Para cada paso en el z-stack, primera exploración con la luz láser que excita inmunofluorescencia de la vGluT-1 anticuerpo para identificar todas las sinapsis glutamatérgicas en un microscopio de campo. El mismo campo se vuelve a escanear a cada paso para FM1-43FX fluorescencia. Establecer filtros de densidad neutra y ganancias fotomultiplicador consistente entre todos los cubreobjetos para un experimento dado, evitando la saturación de la señal. Por lo general adquieren ≥ 5 campos de cada condición en cada experimento. </li><li> Utilizando el software de análisis, convertir la z-stack en un compuesto de un solo plano de la imagen. El proceso que utilizamos para esta conversión produce una imagen basada en la intensidad máxima de cualquier avión en cada píxel. La imagen compuesta es un umbral y un vGluT terminales se identifican para su análisis, sin hacer referencia a la FM1-43FX mancha. Las zonas manchadas, conocido como puntos lagrimales, se marcan como zonas de interés. Por lo general selecciona 10 puntos lagrimales por campo. A continuación, el canal de FM1-43FX es un umbral y las regiones de interés se superponen. El software de análisis se utiliza para cuantificar la intensidad de fluorescencia de cada señal, así como el área de cada punto lagrimal. Un criterio absoluto de píxeles o un criterio del porcentaje de píxeles se puede utilizar para distinguir activa de las sinapsis inactivas. </li></ol> Resultados representante <ol><li> En nuestra cultura, nos encontramos con que el 70-80% de las sinapsis de glutamato, que se define por vGluT-1 manchas, exposición detectable FM1-43 tinción 2, 6-8. En las imágenes superpuestas de color verde fluorescente FM1-43 y el rojo vGluT-1 tinción esto se refleja en una abundancia de color amarillo puntos lagrimales: las sinapsis con las co-localizados marcadores. Estas son las que indican las flechas. </li><li> Aproximadamente el 20-30% de los (rojo) vGluT-1 puntos lagrimales positivo dejar de tener (verde) FM1-43 fluorescencia por encima de línea de base. Estas son las terminales presinápticas inactiva, y un ejemplo es indicado por la flecha. </li><li> Una tercera población de sinapsis también se observó. Estos son positivos para (verde) FM1-43, pero carece de fluorescencia (rojo) vGluT-1 tinción. Estas sinapsis GABAérgicas están activos y un ejemplo es indicado por el asterisco. Estos son excluidos de la mayoría de nuestros análisis, pero puede ser explícitamente estudiada usando un transportador vesicular de anticuerpos GABA en el lugar de la vGluT-1 anticuerpo. </li></ol>

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

Significado <ol><li> Normalmente, las sinapsis se cree que funcionan por la liberación del transmisor con una probabilidad cuantificable. Trabajo por nosotros y los demás se hace evidente que algunas sinapsis son refractarios a la liberación de neurotransmisores, a pesar de una gama completa de transporte vesicular de neurotransmisores y otros indicadores clave presináptica 2, 6-8. Hemos demostrado anteriormente que la actividad de la red básica en las neuronas es fundamental para mantener esta poblaci...

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

Subyace en la plasticidad sináptica probable que la capacidad del sistema nervioso para aprender y recordar y también puede representar una adaptación de otro modo que impide que se convierta en perjudicial insultos neurotóxicos. Hemos estado estudiando una forma de plasticidad presináptica que es interesante, en parte, porque se expresa como una conmutación digital dentro y fuera de la capacidad de un terminal presináptica s para liberar vesículas que contienen el neurotransmisor glutamato. Aquí se demuestra un protocolo para la visualización del estado de la actividad de las terminales presinápticas en cultivos de células disociadas preparado desde el hipocampo de roedores. El método se basa en la detección de las sinapsis activa mediante la tinción con una forma corregible del colorante estirilo FM1-43, comúnmente utilizado para etiquetar las vesículas sinápticas. Este perfil de tinción se compara con la inmunotinción de los mismos terminales con un anticuerpo dirigido contra el transportador de glutamato vesicular 1 (vGluT-1), una mancha diseñado para etiquetar todas las sinapsis de glutamato, independientemente del estado de activación. Nos encontramos con que los estímulos despolarizantes inducir silenciamiento presináptica. La población de sinapsis que no se pronuncia en condiciones basales pueden ser activados por el silenciamiento eléctrico prolongado o por la activación de las vías de señalización del cAMP.

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

Presynaptically Silent Synapses Studied with Light Microscopy

Sinapsis glutamatérgica puede cambiar de un modo activo a un modo silencioso. Se demuestra que el estado de actividad presináptica en la cultura disociada de las neuronas de roedores se visualiza mediante un formulario puede arreglar de la FM1-43 tintes para visualizar las sinapsis activas y inmunotinción con vGluT-1 anticuerpo para visualizar todas las sinapsis de glutamato.

Las sinapsis silenciosa presináptico estudiado con microscopía de luz

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

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

Research

JoVE Journal

Biology

11.4K vistas.  Washington University School of Medicine. Sinapsis glutamatérgica puede cambiar de un modo activo a un modo silencioso. Se demuestra que el estado de actividad presináptica en la cultura disociada de las neuronas de roedores se visualiza mediante un formulario puede arreglar de la FM1-43 tintes para visualizar las sinapsis activas y inmunotinción con vGluT-1 anticuerpo para visualizar todas las sinapsis de glutamato.

Video: Presynaptically Silent Synapses Studied with Light Microscopy

Preparation of Drosophila S2 cells for Light Microscopy

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

Preparation of Drosophila S2 cells for Light Microscopy

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

Preparación de Drosophila Células S2 para microscopía de luz

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

Investigación

Biología

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

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

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

Respuesta de células vivas a la estimulación mecánica estudiados por microscopía óptica integrada la Fuerza Atómica y

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

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

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

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

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

Direct Imaging de calcio ER con Targeted-esterasa inducida colorante de carga (TED)

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

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

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

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

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

El examen de<em&gt; Drosophila</em&gt; Larvas células traqueales de terminales por microscopía de luz

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

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

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

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

Multicapa de montaje para microscopía de luz Hoja de largo plazo del pez cebra

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

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

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

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

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

En Tracking Clonal vivo de madre hematopoyéticas y células progenitoras Marcado por cinco proteínas fluorescentes utilizando confocal y Microscopía Multifotónica

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

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

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

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

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

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

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

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

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

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

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

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

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

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

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

Microscopía de hoja de luz de dinámica cardíaca rápida en embriones de pez cebra

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of&#160;the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software

We present a label-free live imaging protocol using transmitted light microscopy techniques to capture images, analyze and quantify growth kinetics of the filamentous fungus A. nidulans in both submerged cultures and solid media. This protocol can be used in conjunction with fluorescence microscopy.

Análisis cuantitativo de la tasa de crecimiento de Aspergillus nidulans utilizando microscopía en vivo y software de código abierto

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically ...