Questo lavoro è stato supportato da sovvenzioni NIH DA018109 e MH78823, e NIH Neuroscienze Blueprint Nucleo di Grant P30NS057105 alla Washington University.

Cultura preparazione <ol><li> Preparare colture di cellule dissociate di cellule dell&#39;ippocampo di ratto o topo dal primo giorno dopo la nascita 0-3 animali 1. I nostri neuroni aderiscono ad un monostrato di fondo astrociti, che a sua volta aderisce a un livello di collagene diffuso su un vetrino numero 0 spessore. Piastra i neuroni ad una densità di circa 500 cellule per centimetro quadrato. </li><li> Lasciare le culture a crescere per 10-14 giorni per consentire lo sviluppo e la maturazione sinaptica. Trattare le culture in vitro al giorno 4 con il AraC antimitotici di arrestare l&#39;ulteriore crescita gliali. Dar loro da mangiare al giorno in vitro 5 con un cambio medio mezza con media Neurobasal più B27 supplemento per migliorare la sopravvivenza neuronale. </li><li> Durante il periodo di cultura, di introdurre trattamenti mirati a modificare il numero di sinapsi funzionalmente a tacere. Troviamo che un modo comodo per mettere a tacere una larga percentuale (~ 80%) delle sinapsi glutammato è di 4 ore di trattamento con 30 mM di potassio. Più incubazioni con deboli stimoli depolarizzanti induca 2 simili a tacere. Stimolazione di 4 ore con mM forskolin 50, un attivatore di adenilato ciclasi, può essere utilizzato per risvegliare la popolazione di terminali in silenzio al basale 3. </li></ol> Visualizzazione sinapsi silenzioso <ol><li> Le cellule devono avere una densità relativamente bassa, con ben separati i processi neuritiche al giorno 10-14 in vitro in modo da facilitare la visualizzazione di discreto terminali sinaptici usando la microscopia a fluorescenza. </li><li> Preparare una soluzione madre di 5 mm FM1-43FX in acqua distillata. Lo stock dovrebbe essere mantenuta al buio a 4 ° C. Tutti gli esperimenti con FM1-43FX dovrebbe essere eseguita anche al buio. </li><li> Sfida le culture per 2 minuti con 45 mM di cloruro di potassio in soluzione HEPES soluzione tampone contenente 100 mM di cloruro di sodio, cloruro di calcio 2 mM, 1 mM cloruro di magnesio, 10 mM di glucosio e 10 mM FM1-43FX. Questa soluzione deve inoltre contenere 1 micron NBQX e 25 mM D-APV di bloccare i recettori postsinaptici e prevenire le recidive di segnalazione. Questa sfida sarà etichetta terminali sinaptici che sono in grado di esocitosi e endocitosi. La Sfida a tempo e la forza sono progettati per provocare almeno un giro di esocitosi di vescicole tutti disponibili in 4 pool di riciclaggio. Tuttavia, la sfida non è sufficiente a causare il silenziamento supplementare di terminali. </li><li> Lavare la cultura per 3 a 5 secondi solo, utilizzando lo stesso HEPES soluzione tampone senza cloruro di potassio, ma con 500 mM Advasep-7 per rimuovere colorante non specifico 5. Si noti che i tempi di questo passaggio è critico come l&#39;applicazione prolungata di Advasep-7 comporta la perdita di tutti i FM1-43FX etichettatura. Lavaggio successivo a HEPES soluzione tampone senza Advasep-7 per cinque intervalli di 2 minuti. </li><li> Fissare le cellule con paraformaldeide 4% e 0,2% glutaraldeide in PBS, pH 7,4 per 10 minuti. Lavare le cellule una volta brevemente con PBS, poi incubare per 15 minuti in una soluzione bloccante contenente 4% di siero normale di capra e di 0,04% Triton X-100 in PBS. Si noti che FM1-43FX è anche molto sensibile alla quantità di Triton X-100 usato qui e nei passaggi successivi. In generale, FM1-43FX etichettatura è meglio in assenza di Triton X-100, ma alcuni detergenti è necessario permeabilize cellule per immunocolorazione successive. Abbiamo stabilito empiricamente che 0,04% Triton X-100 è ottimale per l&#39;esame delle sinapsi presynaptically silenzio. </li><li> Diluire il primario vGluT-1 anticorpi nel bloccare soluzione ad una concentrazione di 1:2500. Incubare le cellule agitando delicatamente / rotazione nel diluita vGluT-1 anticorpi per 3 ore a temperatura ambiente. Assicurati di coprire i vostri piatti della cultura con un foglio di evitare l&#39;imbiancamento della FM1-43FX colorante. </li><li> Lavare le cellule due volte in PBS e incubare le cellule con un anticorpo anti-guinea maiale coniugato con un fluoroforo con differenti caratteristiche spettrali da FM1-43FX di distinguere le due macchie. Per il nostro esperimento, useremo Alexa 555-coniugato anti-India anticorpi maiale a 1:500 nel bloccare soluzione. Incubare le cellule con l&#39;anticorpo secondario agitando per 30 minuti a temperatura ambiente. </li><li> Lavare le cellule quattro volte in PBS e poi montare il vetrino su un vetrino con una piccola goccia di Fluoromount-G. Assicurarsi di orientare il coprioggetto in modo che le cellule faccia della diapositiva. Lasciare che il vetrini si asciughino durante la notte. </li><li> Siamo ora pronti ad acquisire le immagini delle sinapsi attive e inattive. Preparare un obiettivo olio 60 x 1,4 con un&#39;apertura numerica di un microscopio confocale a scansione laser. Posizionare un vetrino sul tavolo, assicurandosi che il lato coprioggetto sia rivolto verso l&#39;obiettivo. Dopo aver focalizzato la diapositiva, trovare un campo di neuriti, che non è eccessivamente denso. Si raccomanda anche di evitare di imaging in prossimità del soma cellulare, come residuo FM1-43FX colorante può rimanere lì anche dopo il lavaggio estesa. Per le nostre immagini sperimentali, di solito lo zoom per una dimensione di 51 51 micron. </li><li> Acquisireun z-stack di un determinato campo che copre una profondità di 7,8 micron con 27 0,3 micron passi. Questa gamma è sufficiente a coprire lo spessore dei neuriti nella nostra cultura. Per ogni passo nella Z-Stack, prima scansione con la luce laser che eccita immunofluorescenza del vGluT-1 anticorpi per identificare tutte le sinapsi glutammatergica in un campo microscopio. Lo stesso campo viene ri-scansione ad ogni passo per FM1-43FX fluorescenza. Impostare filtri a densità neutra e guadagni fotomoltiplicatore costantemente tra tutti lamelle per un dato esperimento, evitando la saturazione del segnale. Noi di solito acquistano ≥ 5 campi per condizione per ogni esperimento. </li><li> Utilizzando il software di analisi, di convertire lo z-stack in un composto unico piano dell&#39;immagine. Il processo che usiamo per questa conversione produce un&#39;immagine basata sulla massima intensità da qualsiasi piano di ogni pixel. L&#39;immagine composita è thresholded e vGluT-1 terminali sono identificate per l&#39;analisi, senza riferimento alla FM1-43FX macchia. Aree colorate, denominato puncta, sono contrassegnati come regioni di interesse. Noi di solito selezionare 10 puncta per campo. Poi il FM1-43FX canale è thresholded e le regioni di interesse sono sovrapposti. Il software di analisi viene utilizzato per quantificare l&#39;intensità di fluorescenza di ciascun segnale così come l&#39;area di ogni punctum. Un criterio assoluto di pixel o di un criterio percentuale dei pixel può essere utilizzato per distinguere attivo da sinapsi inattive. </li></ol> Rappresentante Risultati <ol><li> Nella nostra cultura, troviamo che il 70-80% delle sinapsi glutammato, definita da vGluT-1 colorazione, mostra rilevabili FM1-43 colorazione 2, 6-8. Nelle immagini sovrapposte di verde FM1-43 e fluorescenza rossa vGluT-1 colorazione si riflette questo come l&#39;abbondanza di giallo puncta: sinapsi con il co-localizzate marcatori. Questi sono indicati dalle frecce. </li><li> Circa il 20-30% di (rosso) vGluT-1 puncta positivo mancherà di avere (verde) FM1-43 fluorescenza di sopra del basale. Questi sono i terminali presinaptici inattivi, e un esempio è indicato dalla freccia. </li><li> Una terza popolazione di sinapsi sarà anche osservato. Questi sono positivi per (verde) FM1-43 fluorescenza, ma privo di (rosso) vGluT-1 colorazione. Queste sinapsi sono attivi GABAergici e un esempio è indicato con l&#39;asterisco. Sono esclusi dalla maggior parte delle nostre analisi, ma può essere esplicitamente studiato utilizzando un anticorpo trasportatore vescicolare del GABA al posto del vGluT-1 anticorpi. </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>

Significato <ol><li> Tipicamente le sinapsi sono pensati per operare attraverso il rilascio di trasmettitore con una probabilità misurabile. Lavoro da noi stessi e gli altri rende evidente che alcune sinapsi sono refrattari al rilascio di neurotrasmettitore, nonostante una serie completa di trasportatore vescicolare dei neurotrasmettitori e altri indicatori chiave presinaptico 2, 6-8. Abbiamo dimostrato in precedenza che l&#39;attività di rete basale nei neuroni è essenziale per mantenere questa popolazi...

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		<th>Material Name</th>
		<th>Type</th>
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		<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

Alla base di plasticità sinaptica probabilmente la capacità del sistema nervoso per imparare e ricordare, che potrebbero anche rappresentare un adattamento che impedisce in qualche modo danneggiare gli insulti di diventare neurotossici. Abbiamo studiato una forma di plasticità presinaptica che è interessante anche perché è espresso in commutazione digitale on e off della capacità di un terminale presinaptico s per rilasciare vescicole contenenti il ​​neurotrasmettitore glutammato. Qui mostriamo un protocollo per visualizzare lo stato di attività dei terminali presinaptici in colture cellulari dissociato preparati con l&#39;ippocampo roditori. Il metodo si basa sulla rilevazione sinapsi attiva utilizzando colorazione con una forma risolvibile del colorante styryl FM1-43, comunemente usato per etichettare vescicole sinaptiche. Questo profilo colorazione viene confrontato con immunocolorazione dei terminali stessi con un anticorpo diretto contro il trasportatore del glutammato vescicolare 1 (vGluT-1), una macchia progettato per etichettare tutti sinapsi glutammato indipendentemente dallo stato di attivazione. Troviamo che stimoli depolarizzanti indurre silenziamento presinaptica. La popolazione di sinapsi che tace in condizioni basali può essere attivato prolungato silenzio elettrico o attraverso l&#39;attivazione di percorsi di cAMP segnalazione.

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

Presynaptically Silent Synapses Studied with Light Microscopy

Sinapsi glutammatergica possibile passare da una modalità attiva di una modalità silenziosa. Abbiamo dimostrato che lo stato presinaptica attività nella cultura dissociata dei neuroni di roditori viene visualizzato per mezzo una forma risolvibile del colorante-43 FM1 per visualizzare sinapsi attiva e immunostaining con vGluT-1 anticorpi per visualizzare tutte le sinapsi glutammato.

Sinapsi Presynaptically silenzioso studiato con microscopia ottica

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 Views.  Washington University School of Medicine. Sinapsi glutammatergica possibile passare da una modalità attiva di una modalità silenziosa. Abbiamo dimostrato che lo stato presinaptica attività nella cultura dissociata dei neuroni di roditori viene visualizzato per mezzo una forma risolvibile del colorante-43 FM1 per visualizzare sinapsi attiva e immunostaining con vGluT-1 anticorpi per visualizzare tutte le sinapsi glutammato.

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.

Preparazione del Drosophila Cellule S2 per la microscopia ottica

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 ...

Ricerca

Biologia

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

Elettrosaldabili cellula visualizzate con microscopia a fluorescenza

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

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.

Risposta cellulare allo stimolo meccanico vivere Studiato da Integrated ottica e microscopia a forza atomica

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.

Imaging diretta di calcio ER con Targeted-esterasi Induced Loading Dye (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.

Esame della<em&gt; Drosophila</em&gt; Larvali tracheali Terminal cellule al microscopio ottico

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.

Multistrato di montaggio per lungo termine foglio leggero Microscopia di Zebrafish

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 clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine ​​fluorescenti che utilizzano confocale e microscopia Multiphoton

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 inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

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.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 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.