Ce travail a été soutenu par des subventions du NIH et de DA018109 MH78823, et les NIH Neuroscience Blueprint base Grant P30NS057105 à l&#39;Université de Washington.

Préparation de la Culture <ol><li> Préparer des cultures de cellules dissociées de cellules de rat ou la souris hippocampe du jour postnatal 0-3 animaux 1. Nos neurones adhèrent à une monocouche sous-jacentes des astrocytes, qui adhère à son tour à une couche de collagène étalé sur une lamelle d&#39;épaisseur numéro 0. Assiette des neurones à une densité d&#39;environ 500 cellules par centimètre carré. </li><li> Autoriser les cultures de croître pendant 10-14 jours pour permettre le développement et la maturation synaptique. Traiter les cultures à jour in vitro, 4 avec l&#39;AraC antimitotiques pour arrêter la croissance d&#39;autres cellules gliales. Nourrissez-les à jour in vitro, 5 avec une moyenne d&#39;échange moitié avec milieu Neurobasal ainsi B27 supplément pour améliorer la survie neuronale. </li><li> Pendant la période de culture, d&#39;introduire des traitements conçus pour modifier le nombre de synapses fonctionnelles réduites au silence. Nous constatons qu&#39;un moyen pratique de réduire au silence un grand pourcentage (~ 80%) des synapses glutamate est un traitement de 4 heures avec 30 mM de potassium. Plus faibles incubations avec des stimuli dépolarisants induisent 2 taire similaires. La stimulation de 4 heures avec 50 uM, un activateur de la forskoline cyclases cyclase, peut être utilisé pour éveiller la population de terminaux silencieuse au départ 3. </li></ol> Visualisation des synapses silencieuses <ol><li> Les cellules doivent avoir une densité relativement faible avec les processus neuritique bien séparés à jour in vitro de 10 à 14 afin de faciliter la visualisation des terminaisons synaptiques discrets en utilisant la microscopie à fluorescence. </li><li> Préparer une solution mère de 5 mM FM1-43FX dans l&#39;eau distillée. Le stock doit être maintenu dans l&#39;obscurité à 4 ° C. Toutes les expériences utilisant FM1-43FX doit également être effectué dans l&#39;obscurité. </li><li> Défi des cultures pendant 2 minutes avec 45 mM de chlorure de potassium dans une solution saline tamponnée HEPES contenant 100 mM de chlorure de sodium, chlorure de calcium 2 mM, 1 mM de chlorure de magnésium, 10 mM de glucose et 10 uM FM1-43FX. Cette solution devrait également contenir une pM et 25 pM NBQX D-APV pour bloquer les récepteurs post-synaptiques et d&#39;empêcher la signalisation récurrentes. Ce défi sera d&#39;étiquettes terminaisons synaptiques qui sont capables de l&#39;exocytose et l&#39;endocytose. Le temps de défi et de résistance sont conçus pour provoquer au moins un tour de l&#39;exocytose des vésicules de tous disponibles dans le pool de quatre recyclage. Cependant, le défi n&#39;est pas suffisante pour causer taire supplémentaire de terminaux. </li><li> Laver la culture pendant 3 à 5 secondes seulement, en utilisant le même HEPES solution saline tamponnée sans chlorure de potassium, mais avec 500 uM Advasep-7 à éliminer le colorant non spécifique 5. Notez que le calendrier de cette étape est essentielle comme l&#39;application prolongée de Advasep-7 se traduira par la perte de tout étiquetage FM1-43FX. Prochain lavage dans une solution saline tamponnée HEPES sans Advasep-7 pendant cinq intervalles de 2 minutes. </li><li> Fixer les cellules avec du paraformaldéhyde 4% et 0,2% de glutaraldéhyde dans du PBS, pH 7,4 pendant 10 minutes. Laver les cellules une fois brièvement avec du PBS, puis incuber pendant 15 minutes dans une solution de blocage contenant 4% de sérum de chèvre normal et 0,04% de Triton X-100 dans le PBS. Notez que FM1-43FX est également très sensible à la quantité de Triton X-100 utilisé ici et dans les étapes ultérieures. De manière générale, FM1-43FX étiquetage est le mieux en l&#39;absence de toute Triton X-100, mais un peu de détergent est nécessaire de perméabiliser les cellules pour l&#39;immunocoloration ultérieures. Nous avons déterminé empiriquement que 0,04% de Triton X-100 est optimale pour l&#39;examen des synapses présynaptique silencieux. </li><li> Diluer le principal vGluT-1 anticorps dans la solution de blocage à une concentration de 1:2500. Incuber les cellules avec agitation douce / rotation dans le diluée vGluT-1 anticorps pour 3 heures à température ambiante. Soyez sûr de couvrir vos boîtes de culture avec du papier pour empêcher le blanchiment de la teinture FM1-43FX. </li><li> Laver les cellules deux fois en PBS puis incuber les cellules avec un anticorps anti-cochon Guinée conjugué à un fluorophore avec des caractéristiques spectrales différentes à partir FM1-43FX de distinguer les deux taches. Pour notre expérimentation, nous allons utiliser Alexa 555-anticorps conjugué de porc anti-Guinée à 1:500 dans une solution de blocage. Incuber les cellules avec l&#39;anticorps secondaire tout en agitant pendant 30 minutes à température ambiante. </li><li> Laver les cellules quatre fois plus dans le PBS et ensuite monter la lamelle sur une lame de verre en utilisant une petite goutte de Fluoromount-G. Veillez à orienter la lamelle afin que les cellules face à la diapositive. Laisser les lames sécher pendant la nuit. </li><li> Nous sommes maintenant prêts pour acquérir des images des synapses actives et inactives. Préparer un objectif d&#39;huile de 60 x 1,4 avec une ouverture numérique sur un microscope confocal à balayage laser. Placer une lame sur la platine, en s&#39;assurant que le côté face de la lamelle est objectif. Après s&#39;être concentrée la diapositive, trouver un domaine de neurites qui n&#39;est pas trop dense. Il est également recommandé d&#39;éviter d&#39;imagerie à proximité du corps cellulaire, comme résiduel FM1-43FX colorant peut y rester même après lavage approfondi. Pour nos images expérimentales, nous avons généralement un zoom sur un champ de 51 51 microns. </li><li> D&#39;acquérirun z-stack d&#39;un domaine donné couvrant une profondeur de 7,8 microns avec 27 0,3 microns étapes. Cette gamme est suffisant pour couvrir l&#39;épaisseur des neurites dans notre culture. Pour chaque étape de la Z-stack, premier scan avec la lumière laser qui excite immunofluorescence de la vGluT-1 anticorps pour identifier toutes les synapses glutamatergiques dans un champ de microscope. Le même champ est re-scanné à chaque étape pour FM1-43FX fluorescence. Set de filtrage de densité neutre et les gains photomultiplicateur constamment entre tous lamelles pour une expérience donnée, tout en évitant la saturation du signal. Nous généralement acquérir ≥ 5 champs par condition pour chaque expérience. </li><li> Utiliser des logiciels d&#39;analyse, de convertir le z-stack dans un composite unique plan de l&#39;image. Le processus que nous utilisons pour cette conversion produit une image basée sur l&#39;intensité maximale de tout avion à chaque pixel. L&#39;image composite est seuillée et vGluT-1 terminaux sont identifiés pour l&#39;analyse, sans référence à l&#39;FM1-43FX tache. Les endroits souillés, dénommé lacrymaux, sont marqués comme des régions d&#39;intérêt. Nous généralement sélectionner 10 points lacrymaux par champ. Suivant le canal FM1-43FX est seuillé et les régions d&#39;intérêt sont superposées. Le logiciel d&#39;analyse est utilisée pour quantifier l&#39;intensité de fluorescence de chaque signal ainsi que la superficie de chaque punctum. Un critère de pixel absolues ou un critère pixel pourcentage peut être utilisé pour distinguer des synapses actives inactives. </li></ol> Les résultats représentatifs <ol><li> Dans nos cultures, nous constatons que 70-80% des synapses du glutamate, défini par vGluT-1 coloration, présentent détectable FM1-43 coloration 2, 6-8. En images superposées de vert FM1-43 de fluorescence rouge et vGluT-1 coloration cela se reflète aussi une abondance de jaune lacrymaux: synapses avec les co-localisés marqueurs. Ils sont indiqués par des flèches. </li><li> Environ 20-30% des (en rouge) vGluT-1 lacrymaux positive manquer d&#39;avoir (en vert) FM1-43 fluorescence au-dessus de base. Ce sont les terminaux inactifs présynaptique, et un exemple est indiqué par la flèche. </li><li> Une troisième population des synapses seront également observés. Ce sont positifs pour (vert) FM1-43 de fluorescence, mais dépourvue de (rouge) vGluT-1 coloration. Ce sont des actifs synapses GABAergiques et un exemple est indiqué par l&#39;astérisque. Ce sont exclus de la plupart de nos analyses, mais peut être explicitement étudiées en utilisant un anticorps transporteur vésiculaire du GABA à la place du vGluT-1 anticorps. </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>

Signification <ol><li> Typiquement synapses sont pensés pour fonctionner en libérant l&#39;émetteur avec une probabilité mesurable. Le travail par nous-mêmes et d&#39;autres, il est évident que certaines synapses sont réfractaires à la libération de neurotransmetteurs, en dépit d&#39;une gamme complète de transporteur vésiculaire des neurotransmetteurs et des autres principaux marqueurs présynaptique 2, 6-8. Nous avons montré précédemment que l&#39;activité du réseau basale dans les neuron...

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

Sous-tend la plasticité synaptique vraisemblablement la capacité du système nerveux, d&#39;apprendre et de se souvenir et peut également représenter une adaptabilité qui empêche par ailleurs d&#39;endommager les insultes de devenir neurotoxiques. Nous avons étudié une forme de plasticité présynaptique qui est intéressant, en partie parce qu&#39;elle est exprimée comme une commutation numérique sur et en dehors de la capacité d&#39;un terminal présynaptique s pour libérer des vésicules contenant le neurotransmetteur glutamate. Ici nous démontrons un protocole pour la visualisation de l&#39;état d&#39;activité des terminaisons présynaptiques dans les cultures cellulaires dissociées préparées à partir de l&#39;hippocampe chez les rongeurs. La méthode repose sur la détection des synapses actives utilisant une coloration avec une forme fixable du colorant styryle FM1-43, communément utilisé pour étiqueter les vésicules synaptiques. Ce profil est comparé à coloration immunologique des bornes même avec un anticorps dirigé contre le transporteur du glutamate vésiculaire 1 (vGluT-1), une tache conçue pour étiqueter toutes les synapses glutamate indépendamment de leur statut d&#39;activation. Nous constatons que les stimuli dépolarisants induisent taire présynaptique. La population de synapses qui est silencieux dans des conditions de base peuvent être activés en faisant taire électriques prolongées ou par l&#39;activation des voies de signalisation de l&#39;AMPc.

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

Presynaptically Silent Synapses Studied with Light Microscopy

Synapses glutamatergiques peut basculer d&#39;un mode actif à un mode silencieux. Nous démontrons que le statut de l&#39;activité présynaptique dans la culture de neurones dissociés de rongeurs est visualisée en utilisant un formulaire fixable du FM1-43 de colorant pour visualiser synapses actives et immunomarquage avec des anticorps vGluT-1 pour visualiser toutes les synapses glutamate.

Synapses présynaptique silencieux étudiée en microscopie optique

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 vues.  Washington University School of Medicine. Synapses glutamatergiques peut basculer d'un mode actif à un mode silencieux. Nous démontrons que le statut de l'activité présynaptique dans la culture de neurones dissociés de rongeurs est visualisée en utilisant un formulaire fixable du FM1-43 de colorant pour visualiser synapses actives et immunomarquage avec des anticorps vGluT-1 pour visualiser toutes les synapses glutamate.

Vidéo: 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.

Préparation des Drosophile Les cellules S2 pour la microscopie optique

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 ...

Recherche

Biologie

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.

Réponse Live Cell à la stimulation mécanique étudiée par microscopie à force atomique et optique intégré

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.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (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.

Examen de<em&gt; Drosophila</em&gt; Larvaires cellules terminales trachéales par microscopie optique

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.

Multicouche de montage pour à long terme Fiche microscopie optique de poisson zèbre

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

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

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

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

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

In vivo suivi clonale de cellules souches hématopoïétiques et progénitrices Marqué par cinq protéines fluorescentes confocale et multiphotonique Microscopie

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

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

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

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

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

In Vivo suivi 4-Dimensional de hématopoïétiques cellules souches et progénitrices adultes dans la voûte du crâne de souris de moelle osseuse

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.

Analyse du Gap Junction-dependent Transfert de miARN avec microscopie 3D-FRAP

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.

Microscopie à feuille de lumière de la dynamique cardiaque rapide chez les embryons de poisson zèbre

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.

Analyse quantitative du taux de croissance d’Aspergillus nidulans à l’aide de la microscopie en direct et d’un logiciel open source

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