Name: two-photon-based photoactivation in live zebrafish embryos
Uploaded: 2010-12-24T15:00:00
Description: Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Tack till Genia Brodsky för figur grafik, Vyacheslav Kalchenko, Douglas Lutz och Leonid Roitman för teknisk rådgivning och hjälp med de två-photon uncaging, Maayan Tahor och Suliman Elsadin för tekniskt stöd, Uwe Stråhle för vänligt ge neurogenin1 reportern linje och Amos Gutnick för kommentarer till detta manuskript. Forskningen inom Levkowitz labbet stöds av den tysk-israeliska Foundation (licensnummer 183/2007), Israel Science Foundation (licensnummer 928/08) och Harriet &amp; Marcel Dekker Foundation. GL är en sittande i Tauro Career Development professur i biomedicinsk forskning.

Vi beskriver ett protokoll för cell-märkning med bur fluorescein kan dock andra foto-aktiveras genom färgämnen och proteiner på liknande sätt användas. 1. Injektion av Caged Fluorescein <ol><li> Bered 5% stamlösning (5 mg bur-fluorescein / 100 mikroliter 0,2 KCl) av dextran-konjugerad 4,5-dimetoxi-2-Nitrobenzyl (DMNB) bur fluorescein (10.000 MW dextran, anjoniska, Invitrogen, molekylära sonder, Karlsbad, Kalifornien , kat. nej. D-3310). Fördela och förvara i -20 ° C. Observera ...

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S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale genetically targeted optical control of neural activity.</a> Nat Neurosci. 8, 1263-1268 (2005).</li><li>Bulina, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromophore-assisted+light+inactivation+(CALI)+using+the+phototoxic+fluorescent+protein+KillerRed.">Chromophore-assisted light inactivation (CALI) using the phototoxic fluorescent protein KillerRed.</a> Nat Protoc. 1, 947-9453 (2006).</li><li>Adams, S. R., Tsien, R. 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Foto-aktiveras genom föreningar är molekyler vars funktion är maskerad tills de belyses med en viss våglängd (oftast UV), förmå en fotokemisk reaktion som omvandlar molekyler i ett biologiskt eller kemiskt aktivt läge. Dessa sonder ger mycket kraftfulla verktyg i cellbiologi forskning, eftersom aktiveringen kan justeras tidsmässigt och rumsligt genom att begränsa sin exponering för ljus. Den betydande fördel av multi-foton mikroskopi är dess relativt djup optiska inträngning oc...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3310</td>
<td>molecular probes</td>
</tr>
<tr>
<td>Agarose for injection trough and coated plates</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Thin Wall Glass Capillaries with filament</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-6</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Microloader tip</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic picopump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PV820</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>22290-9</td>
<td>&nbsp;</td>
<tr>
<td>Low melting point agarose for embryo mounting</td>
<td>&nbsp;</td>
<td>Ultra Pure LMP agarose</td>
<td>16520100</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>Fast Red</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11496549001</td>
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two-photon-based photoactivation in live zebrafish embryos

Fotoaktivering av mål föreningar i en levande organism har visat sig vara en värdefull metod för att undersöka olika biologiska processer som embryonal utveckling, cellulär signalering och vuxna fysiologi. I detta avseende möjliggör användning av multi-foton mikroskopi kvantitativa fotoaktivering av ett visst ljus lyhörd agent i djupa vävnader i en enda cell upplösning. Som zebrafisk embryon är optiskt transparenta, kan deras utveckling följas in vivo. Dessa egenskaper gör zebrafisk en perfekt modell organism för att kontrollera aktiviteten hos en mängd olika kemiska ämnen och proteiner med fokuserat ljus. Här beskriver vi användandet av två-photon mikroskopi för att framkalla en aktivering av kemiskt bur fluorescein, vilket i sin tur ger oss möjlighet att följa cellens öde i levande zebrafisk embryon. Vi använder embryon som uttrycker en levande genetisk landmärke (GFP) för att lokalisera och exakt rikta alla celler av intresse. Detta förfarande kan vara lika användas för exakta ljus inducerad aktivering av proteiner, hormoner, små molekyler och andra bur föreningar.

Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Multiphoton mikroskopi tillåter kontroll av låg energi fotoner med djup optisk penetration och minskad fototoxicitet. Vi beskriver användningen av denna teknik för levande cell märkning i zebrafisk embryon. Detta protokoll kan lätt anpassas för foto-induktion av olika ljus-känslig molekyler.

Två-foton-Based Fotoaktiveringen i Live Zebrafish embryon

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

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Mikroinjektion av mRNA och Morpholino oligonukleotider Antisens i Zebrafish embryon.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

Mikroinjektion av Zebrafish embryon till Analysera genfunktion

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

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Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

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Bildproduktion för cell motilitet och Actin cytoskelettet av enskilda nervceller och neurala celler Crest i Zebrafish embryon

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

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Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans i Zebrafish embryon genom Metabola märkning och Bioorthogonal Klicka Kemi

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

Metabolisk profil Analys av zebrafisk embryon

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Facial Transplants in Xenopus laevis Embryos

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Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Analysis of Oxidative Stress in Zebrafish Embryos

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High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...