Ce travail a été soutenu par les US National Institutes of Health Grant NS-024572 (à RSW).

1. Colorer la préparation de colorants supravital <ol><li> Pour le protocole de dissection des couleuvres voir Stewart et al. 5 et Teng et al. 6 tissus reptile reste physiologique pour des durées plus longues, et à la contamination bactérienne moins, lorsqu&#39;il est conservé à basse température (voir ci-dessous). Un tissu de mammifère est généralement maintenu à la température ambiante ou plus élevée. </li><li> Pour lysosomale vital coloration de colorant, dissoudre le colorant dans reptiles 1:5000 de solution de Ringer froid (0,2 pm). Incuber à 4 ° C pendant 15 min. Lavez plusieurs fois avec Ringer froid. Image dès que possible. </li><li> Pour FM1-43 coloration à l&#39;aide de KCl stimulation, diluer le colorant en haute KCl Ringers 1:500 (7 M). Incuber à 4 ° C pendant 30 à 60 secondes maximum. Laver rapidement trois fois avec Ringer froid, ~ 1 min / rinçage. Image dès que possible. </li><li> Pour FM1-43 coloration utilisant hypertonique (saccharose) stimulation, diluer le colorant dans 0,5 M sonneries de saccharose comme ci-dessus. Incuber à 4 &amp; #176; C pendant 2-5 min. Laver comme à l&#39;étape 1.3 ci-dessus avec Ringer froid. Image dès que possible. </li><li> Pour FM1-43 coloration par stimulation électrique, voir Teng et al. 6 En bref, la préparation est placé un plat contenant du sérum physiologique et le colorant. Le nerf est stimulé par un train préprogrammée de 200 microsecondes impulsions rectangulaires (~ 7 V, 30 Hz, 18 ps). Laver comme à l&#39;étape 1.3 ci-dessus avec Ringer froid. Image dès que possible. </li></ol> 2. Configurez la préparation pour l&#39;imagerie <ol><li> Orienter la préparation de telle sorte que les structures d&#39;intérêt sont aussi proches que possible de l&#39;objectif du microscope. </li><li> Si un microscope inversé est utilisé, utiliser une chambre dont le fond contient une fine lamelle ronde. En règle générale, la chambre de formation d&#39;image doit avoir un fond en acier inoxydable mince avec un diamètre de 25 mm # 1 en verre d&#39;épaisseur lamelle au centre. Si un microscope droit est utilisée, une lamelle de fond n&#39;est pas nécessaire mais elle est utilepour permettre l&#39;imagerie de la lumière transmise (par exemple de contraste d&#39;interférence différentiel, DIC) pour orienter l&#39;échantillon et de localiser des objets d&#39;intérêt. </li><li> Choisissez un objectif approprié pour obtenir la résolution la plus élevée possible 3D. Il ya des compromis entre ouverture numérique (NA), la distance de travail, agrandissement, et le type de lentille (sec, immersion dans l&#39;huile à l&#39;eau et). Pour les préparations minces comme des cultures de tissus, les objectifs de pétrole sont les meilleurs. En utilisant un microscope droit, flotter une lamelle sur le bain aqueux, et utiliser de l&#39;huile d&#39;immersion entre le sommet de la lamelle et l&#39;objectif. </li><li> Si le logiciel de déconvolution est utilisé, le vendeur peut fournir des fonctions d&#39;étalement de point prédéterminé (PSF) de certains objectifs. Si cette information n&#39;est pas disponible, ou si un objectif pris en charge est choisie, déterminer la PSF par imagerie micro-pointes fluorescentes ou des objets de diffraction limitée similaires 7,8. </li></ol> 3. Établir la profondeur de champ désirée et le nombre de Images souhaités par Time-lapse cadre <ol><li> Sélectionnez la profondeur totale du champ de sorte que le déplacement ou l&#39;interaction des structures d&#39;intérêt ne disparaissent pas ou sortir de concentration qui se déplacent dans la direction z. Le champ de z doit légèrement dépasser la dimension verticale de la procédure de cellule ou cellule étant imagée. Pour les bornes du moteur vertébrés, 15-20 um est typique. </li><li> Calculer le pas de l&#39;axe z nécessaire pour chaque incrément de mise au point. Résolution le long de l&#39;axe des z varie en fonction des propriétés de l&#39;objectif; il est d&#39;environ un quart de la résolution du plan xy. Si l&#39;imagerie confocale ou un logiciel de déconvolution est utilisé, la résolution de l&#39;axe z est améliorée. Améliorer la résolution finale par suréchantillonnage délibéré dans la direction z 5, mais aussi voir l&#39;étape 3.3 ci-dessous. Un intervalle typique de l&#39;étape est dans la plage de 400-1,500 nm pour les objectifs de 40 100X. La lumière doit être coffré off entre chaque image z-étape. </li><li> Sélectionnez le nombre d&#39;images par z-pile, à savoir la profondeur de champ souhaitée diviséepar l&#39;intervalle de l&#39;étape. Temps requis pour remplir un z-stack typique est de 1-10 sec. Objets se déplaçant rapidement (100 nm / s) peuvent apparaître floue dans une pile d&#39;images 3D car chaque plan image correspond à un point de temps légèrement différente. En outre, chaque image supplémentaire contribue à photoblanchiment et de phototoxicité possible (voir Discussion). Si les deux cas concerne, choisir un z-étape plus grande pour recueillir moins d&#39;images par pile. Compenser plus tard en utilisant un logiciel d&#39;interpolation d&#39;image (étape 6.3 ci-dessous). </li></ol> 4. Sélectionnez le Frame Rate Time-lapse Choisissez par l&#39;expérimentation un taux qui est juste suffisant pour résoudre en douceur les changements avec le temps comme le mouvement, les interactions ou les événements de fusion 9. Sous échantillonnage peut produire des artefacts de mouvement (erreurs d&#39;échantillonnage), tandis que le suréchantillonnage augmente inutilement l&#39;exposition à la lumière. Collecte, soit une longue séquence unique ou plusieurs séquences répétées de la même préparation, chacun avec un relativement faible intervalle de temps totale ( <em&gt; par exemple 10 points de temps x 30 sec d&#39;intervalle = 5 min). Si possible, voir la préparation avec un éclairage à épifluorescence continue à estimer grossièrement les vitesses de déplacement, y compris la dérive de l&#39;ensemble de la préparation si elle est présente. 5. Remplissez tous Imaging Live pour une préparation particulière Préparations reptiles restent généralement robuste pour ~ 1-2 heures, plus si on le refroidit. 6. Analyser les données après utilisation désiré <ol><li> Conservez tous les fichiers de données brutes. Tandis que le stockage numérique n&#39;est généralement pas un problème, le temps de traitement est important, même avec des ordinateurs ou postes de travail personnels rapides. Pour cette raison, les images des cultures dans une région d&#39;intérêt [ROI]. Assurez-vous que l&#39;ensemble de la région d&#39;intérêt est dans la fenêtre recadrée au cours du temps ensemble. </li><li> Deconvolve piles d&#39;images en utilisant la fonction correcte propagation point un logiciel approprié et. Assurez-vous que la résolution est améliorée et qu&#39;aucun artefact a été générée par lealgorithme de déconvolution. Figure 3 montre des exemples d&#39;images. </li><li> Utiliser un algorithme d&#39;interpolation d&#39;étendre la résolution apparente de l&#39;axe z. Une expansion 6X à partir d&#39;un plan réel séparation d&#39;image de 1,5 um à une apparente 0,25 um séparation est suggéré. Vous pouvez également utiliser une combinaison de suréchantillonnage (étape 3.2) et l&#39;interpolation. </li><li> Effectuer le contraste, la luminosité, le filtrage de bruit, photoblanchiment et autres ajustements typiques de traitement d&#39;image, si désiré. normes de manipulation de l&#39;image dictent que pour la plupart des travaux scientifiques, il convient que les mêmes corrections être appliquées à toutes les images, à la fois dans une pile et à différents moments. La conséquence d&#39;un réglage particulier devrait être évaluée chez toutes les images 9. </li><li> Sélectionnez un mode d&#39;affichage particulier pour l&#39;exportation de données. L&#39;affichage est plus simple vidéo stéréo en utilisant soit des anaglyphes rouge-vert ou rouge-bleu (exemples de la figure 3), des paires stéréo, ou en alternance gauche / droit œil cadres avec des lunettes à obturateur. Anaglyphes sont limités à une seule couleur. Améliorer la profondeur apparente de l&#39;image si l&#39;on souhaite améliorer la résolution visuelle de l&#39;axe z. </li><li> Expérimenter diverses techniques d&#39;amélioration de filtrage et de l&#39;image. Par exemple, le filtrage de Laplace est utile pour augmenter le contraste des petites structures ci-dessus fond. La luminosité des pixels au centre de la fenêtre courante est améliorée tandis que la luminosité des zones environnantes est réduite. Notez que certaines méthodes de filtrage ne fonctionnent pas bien en combinaison. </li><li> Appliquer la correction de dérive si il ya un mouvement important de la préparation au fil du temps. Un tel mouvement est commun dans la vie muscle, même avec des médicaments ajoutés à supprimer des potentiels d&#39;action et potentiels synaptiques. Un algorithme d&#39;enregistrement de pixels (par exemple. D&#39;Imaris, Adobe After Effects, le plugin pour ImageJ TurboReg) aligne les images en accéléré et peut réduire, mais généralement pas éliminer complètement, telle motion. </li></ol>

<ol>
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Les auteurs déclarent aucun intérêt financier concurrents.

L&#39;aspect le plus critique de l&#39;imagerie 4D est la gestion de la durée et de l&#39;intensité de l&#39;exposition de la lumière. Photoblanchiment d&#39;image diminue le rapport signal-sur-bruit et peut être problématique ou non en fonction de divers facteurs, y compris le choix des fluorophores. Non spécifique des dommages aux tissus vivants (de phototoxicité) est liée au photoblanchiment, et peut parfois être identifiée en utilisant des sondes fluorescentes conçus à cet effet ou 2,12 par ex...

Imagerie time-lapse de tissu vivant offre un accès visuel à dynamiques des relations structure-fonction qui ne peut être apprécié dans les préparations fixes ou vivant imagées en un seul point dans le temps. Souvent, cependant, l&#39;arbitrage pour l&#39;accès à l&#39;information temporelle est une diminution de la résolution optique. Ouverture numérique des objectifs élevés huile d&#39;immersion ne sont pas pratiques dans les tissus vivants en raison de leur gamme étroite de mise au point, laissant immersion dans l&#39;eau ou les objectifs secs comme les seules alternatives. En outre, la résolution accrue offerte par l&#39;optique confocale ne peut pas être utilisée dans certaines préparations de vie en raison de la phototoxicité des niveaux relativement élevés d&#39;éclairement requis de 1,2. Enfin, alors que plusieurs techniques optiques en temps réel ou en accéléré sont disponibles qui offrent résolution améliorée, leur applicabilité est limitée aux préparations où les structures d&#39;intérêt peuvent être positionnés à quelques centaines de nanomètres de l&#39;objectif 1. La méthode décriterend l&#39;utilisation de l&#39;équipement relativement peu coûteux, est polyvalent, tout en offrant une meilleure résolution par rapport aux techniques time-lapse plus couramment utilisés. Il est destiné à être utilisé dans les laboratoires individuels ainsi que des installations d&#39;imagerie. Le procédé utilise la microscopie à épifluorescence classique, combiné avec un appareil photo numérique sensible et avec du matériel destiné à acquérir rapidement des ensembles d&#39;images légèrement différentes plans focaux (z-piles). Chaque z-stack est numériquement deconvolved pour augmenter la résolution. Une caractéristique de la 3D time-lapse (4D) imagerie est suivi précis des organites ou d&#39;autres structures en mouvement. Lorsqu&#39;il est correctement mis en place, les structures imagées ne sortent pas de mise au point, et le mouvement dans les trois directions peuvent être observés et quantifiés. Ainsi, il est impossible pour une structure teinté à disparaître sur un ou plusieurs cadres time-lapse seulement par la dérive au-dessus ou en dessous d&#39;un plan focal étroit. La méthode est aussi un outil sensible pour évaluer les interactions et possible fusion de petites structures. Épifluorescence classique ou images confocales de structures proches de la limite de diffraction (quelques centaines de nm) ne confirment pas la fusion même si les images fusionnées montrent chevauchement de leurs étiquettes respectives 3. Fusion est suggéré, mais il reste possible que les objets sont séparés horizontalement et verticalement par une distance qui est inférieure à la limite de diffraction. Trois ou à quatre dimensions imagerie, en revanche, permet la visualisation des objets dans chacune des trois directions orthogonales. L&#39;apparition de la fusion dans les trois vues augmente le niveau de sécurité. Et, dans certaines préparations de vie, la direction ou le mouvement brownien des objets présumément fusionnés fournit une preuve supplémentaire lorsque les deux étiquettes se déplacent ensemble dans le temps. Bien sûr, quand près de la limite de diffraction du niveau de certitude dans les structures les plus exigeants de fond, ou montrant qu&#39;ils contiennent deux colorants (fusion), n&#39;est pas absolue. Le cas échéant, des techniques spécialisées, telles que le transfert d&#39;énergie par résonance de fluorescence (FRET)4, sont plus appropriées.

<table border="0" cellpadding="0" cellspacing="0" style="width:720px;" width="720">
	<colgroup>
		<col span="4" />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:42px;width:180px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:180px;">Catalog Number</td>
			<td style="width:180px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SGC5</td>
			<td>Biotium [Hayward, CA]</td>
			<td>70057</td>
			<td>Final conc:10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM1-43FX</td>
			<td>Invitrogen [Carlsbad, CA]</td>
			<td>F35335</td>
			<td>Final conc:7 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LysoTracker Red</td>
			<td>Invitrogen [Carlsbad, CA]</td>
			<td>L7528</td>
			<td>Final conc:0.2 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Solutions:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reptilian Ringers pH 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>145 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>2.5&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td style="width:180px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:21px;">High KCL Reptilian Ringers pH 7.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>&#160;86&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>&#160;60&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:21px;">High Sucrose Ringers pH 7.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>145 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>2. 5 mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">CaCl2</td>
			<td>3.6&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">MgSO4</td>
			<td>1.8&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">KH2PO4 (Dibasic)</td>
			<td>1.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>5.0&#160; mM</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sucrose</td>
			<td>0.5 M (17.1 gm/50 mL)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Comments</td>
			<td>Comments(website)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Axioplan 200 inverted microscope</td>
			<td colspan="2">Carl Zeiss [Thornwood, NY]</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">N-Achroplan 63X water objective; n.a.=0.9; Working distance=2.4mm&#160;</td>
			<td colspan="2">Carl Zeiss [Thornwood, NY]</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">DG4 combination light source/excitation filterwheel switcher</td>
			<td>Sutter instruments [Novato, CA]</td>
			<td>175W Xenon arc lamp</td>
			<td><a href="http://www.sutter.com/">www.sutter.com</a></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Lambda 10-2&#160; emission filterwheel switcher</td>
			<td colspan="2">Sutter instruments [Novato, CA]</td>
			<td><a href="http://www.sutter.com/">www.sutter.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sensicam CCD camera</td>
			<td colspan="2">Cooke Instruments [Tonawanda, NY]</td>
			<td><a href="http://www.cookecorp.com/">www.cookecorp.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cascade 512 CCD camera</td>
			<td colspan="2">Photometrics [Tucson, AZ]</td>
			<td><a href="http://www.photometrics.com/">www.photometrics.com</a></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">Imaging dishes- made in-house-11cm dia.; 25 mm dia. #1 coverslip embedded; magnetic pins</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Comments</td>
			<td>Comments(website)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slidebook 5.0</td>
			<td>Intelligent Imaging Innovations [Denver, CO]</td>
			<td>Deconvolution; Drift correction;3D and 4D data presentation</td>
			<td><a href="http://www.intelligent-imaging.com/">www.intelligent-imaging.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IMARIS 7.5.2</td>
			<td>Bitplane [South Windsor, CT]</td>
			<td>Drift correction; 3D and 4D data presentation</td>
			<td><a href="http://www.bitplane.com/">www.bitplane.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AfterEffects CS6</td>
			<td>Adobe [San Jose, CA]</td>
			<td>Drift correction</td>
			<td><a href="http://www.adobe.com/">www.adobe.com</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ 1.46</td>
			<td>National Institutes of Health [Bethesda, MD]</td>
			<td>Multiple plugins available;Stereo pair construction</td>
			<td><a href="http://rsbweb.nih.gov/ij">http://rsbweb.nih.gov/ij</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss LSM</td>
			<td>Carl Zeiss [Thornwood, NY]</td>
			<td>Stereo pair construction</td>
			<td><a href="http://www.zeiss.com/">www.zeiss.com</a></td>
		</tr>
	</tbody>
</table>

visualization of endosome dynamics in living nerve terminals with four-dimensional fluorescence imaging

Quatre dimensions (4D) Imagerie de la lumière a été utilisé pour étudier le comportement des petites structures au sein terminaisons nerveuses motrices de la transverse de l&#39;abdomen mince muscle de la couleuvre rayée. Les données brutes comprend des séquences de time-lapse de 3D z-piles. Chaque pile contient 4-20 images acquises avec une optique à épifluorescence à plans focaux séparés par 400-1,500 nm. Les étapes de l&#39;acquisition de piles d&#39;images, telles que le réglage de mise au point, la commutation de longueurs d&#39;onde d&#39;excitation, et le fonctionnement de l&#39;appareil photo numérique, sont automatisées autant que possible pour maximiser le taux d&#39;image et de minimiser les dommages aux tissus exposés à la lumière. Après l&#39;acquisition, une série de piles d&#39;images est une déconvolution pour améliorer la résolution spatiale, converties au format 3D désirée, et sert à créer un &quot;film&quot; 4D qui est appropriée pour diverses analyses sur ordinateur, en fonction des données expérimentales recherchées. Une application est l&#39;étude du comportement dynamique de deux classes de endosomes trouvés dans nerveuses bornes-macroendosomes (ME)et endosomes acides (EI)-dont la taille (200-800 nm pour les deux types) sont à ou près de la limite de diffraction. Accès à l&#39;information 3D à chaque point de temps offre plusieurs avantages par rapport à l&#39;imagerie time-lapse classique. En particulier, la taille et la vitesse de déplacement de structures peuvent être quantifiés dans le temps sans perte de netteté. Des exemples de données de l&#39;imagerie 4D montrent que les ME se rapprochent de la membrane plasmique et disparaissent, ce qui suggère qu&#39;ils sont exocytosed plutôt que de déplacer simplement verticalement à une distance à partir d&#39;un seul et même plan de mise au point. Est également révélé fusion putatif de ME et EI, par visualisation de chevauchement entre les deux structures contenant un colorant telles que vues dans chacune des trois projections orthogonales.

Les données présentées sont des terminaux serpent neuromusculaires (voir vues basses et hautes grossissement de la figure 3, le colorant d&#39;endocytose (FM1-43) adoption crée une brume qui remplit chaque bouton) et, en particulier, macroendosomes (ME) et les endosomes acides (EI) à l&#39;intérieur de ces bornes 5. ME sont créés par endocytose vrac au cours de l&#39;activité neuronale 10 et leur nombre diminue de façon exponentielle avec le temps après une activité a c...

Watch this Scientific Journal Video about Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging at JoVE.com

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Quatre dimensions (4D) imagerie est utilisée pour étudier le comportement et les interactions entre les deux types de endosomes dans les terminaisons nerveuses vertébrés vivant. Le mouvement de ces petites structures est caractérisé en trois dimensions, ce qui permet la confirmation des événements tels que la fusion des endosomes et l&#39;exocytose.

Visualisation des Endosome Dynamics dans Vivre terminaisons nerveuses avec quatre dimensions d&#39;imagerie par fluorescence

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

visualization-endosome-dynamics-living-nerve-terminals-with-four

Research

JoVE Journal

Neuroscience

8.9K vues.  Washington University School of Medicine.  Quatre dimensions (4D) imagerie est utilisée pour étudier le comportement et les interactions entre les deux types de endosomes dans les terminaisons nerveuses vertébrés vivant. Le mouvement de ces petites structures est caractérisé en trois dimensions, ce qui permet la confirmation des événements tels que la fusion des endosomes et l'exocytose. 

Vidéo: Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

Préparation de vie isolées cellules photoréceptrices vertébrés pour l&#39;imagerie de fluorescence

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

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Neurosciences

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp mesures de capacité et de Ca 2 + Imagerie à terminaisons nerveuses rétiniennes unique en tranches

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisation des propriocepteurs dans<em&gt; Drosophila</emLes larves&gt; et pupes

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La stimulation cérébrale profonde avec simultanée IRMf chez les rongeurs

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

Dans l&#39;imagerie in vivo de Optic Nerve Fiber intégrité par IRM de contraste amélioré chez la souris

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagerie Ca<sup&gt; 2+</sup&gt; Dynamics dans les terminaux cône photorécepteur Axon de la rétine de souris

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

Surveillance optique de Living Nerve Terminal Marquer follicule pileux lancéolées Endings du<em&gt; Ex Vivo</em&gt; Souris oreille peau

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantification des molécules d&#39;endosome et de lysosome dans les neurones cultivés utilisant des sondes fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Visualization of Amyloid &amp;#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Visualisation des dépôts β-amyloïde dans le cerveau humain avec Laser assistée par matrice désorption-ionisation d’imagerie de la spectrométrie de masse

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Préparation du tissu cérébral nouveau-né du rat pour l’analyse morphométrique ultrastructurale de la distribution synaptique des vésicules aux terminaux nerveux