We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange mRNA in vitro trascrizione e mRNA Purification <ol><li> Linearizzare il H2B-PSmOrange contenente pCS2 + plasmide con l&#39;enzima di restrizione NotI secondo le istruzioni del produttore. Nota: eseguire i passi successivi utilizzando adeguate protezioni quali guanti e camice da laboratorio per evitare la contaminazione mRNA e il degrado. </li><li> Purificare il DNA linearizzato utilizzando un kit di purificazione o di fenolo-cloroformio PCR metodi basati secondo protocolli produttori. </li><li> Utilizzare 1 mg di DNA modello linearizzato per Sp6-mRNA trascrizione secondo le istruzioni del produttore. </li><li> Rimuovere DNA con l&#39;aggiunta di 1,0 U RNasi libero DNasiI per 10-20 minuti a 37 ° C. </li><li> Pulire l&#39;mRNA utilizzando un RNA ripulire kit secondo il protocollo del produttore. </li><li> Per aumentare mRNA purezza e la concentrazione, precipitare l&#39;mRNA aggiungendo 6 microlitri di acetato di sodio (3,0 M, pH 5,2) e 150 microlitri 96% di etanolo. Incubare il msoluzione ixed a -20 ° C per almeno 30 minuti e fino a 24 ore. </li><li> Raccogliere il mRNA facendo girare la soluzione a 18,407 xg per 45 min a 4 ° C. Eliminare il liquido e risospendere il mRNA in 150 ml 70% di etanolo. Mescolare e centrifugare la soluzione per altri 15 min a 4 ° C. Eliminare il liquido, asciugare il pellet per 5 minuti in ghiaccio e risospendere il mRNA in 20 microlitri ultrapura RNasi acqua libera. </li><li> Valutare la concentrazione e la purezza del mRNA mediante misurazione fotometrica di una diluizione 1: 100 di mRNA (1 ml mRNA in 99 microlitri ultrapura RNasi acqua libera). </li><li> Per la conservazione a breve termine, mantenere la soluzione mRNA a -20 ° C. Per la conservazione a lungo termine, mantenere l&#39;mRNA a -80 ° C. </li></ol> 2. H2B-PSmOrange mRNA microiniezione in embrioni di zebrafish <ol><li> Impostare accoppiamento coppie la sera prima iniezione utilizzando tg (foxD3: GFP); tg (FLH: GFP) doppio pesci transgenici (o qualsiasi altro pesce transgenico GFP). Lasciare il pesce separato inserendo un distanziatoretra di essi per controllare il tempo di accoppiamento. </li><li> Rimuovere il distanziale di mattina di iniezione e lasciare che il compagno di pesce per 20 minuti. </li><li> Durante il tempo di accoppiamento, diluire il H2B-PSmOrange mRNA alla concentrazione finale di 130 pg / nl (in RNasi acqua libera) e il trasferimento 6 ml soluzione per l&#39;iniezione in un capillare microiniezione utilizzando una punta microloader. Controllare il volume di iniezione con un vetrino calibrato. Regolare la pressione di iniezione per iniettare 2 soluzione di mRNA nl (260 pg). </li><li> Raccogliere le uova in una piastra di Petri di plastica sterilizzati contenente 1x Embryo (E3) di media. </li><li> Trasferimento da 20 a 30 embrioni per un piatto di iniezione con una pipetta di plastica Pasteur. </li><li> Iniettare 2 mRNA nl contenenti soluzione nella cella o appena sotto la cella nel tuorlo allo stadio unicellulare 11. </li><li> Trasferire gli embrioni iniettato in una piastra di Petri contenente medio 1x E3, rimuovere gli embrioni normalmente in via di sviluppo non fecondate o meno e crescere a 28 ° C. Nota: la protezione della luce of embrioni non è richiesto durante l&#39;esperimento. </li><li> Sollevare embrioni di zebrafish in media 1x E3 e aggiungere 0,2 mM PTU (1-fenil-2 tiourea) dopo gastrulation per inibire la pigmentazione. Cambiare il mezzo due volte al giorno per minimizzare il rischio di contaminazioni batteriche. </li></ol> 3. Embrione Embedding <ol><li> Selezionare la GFP (verde) e PSmOrange (arancione / rosso) embrioni co-esprimono usando un microscopio binoculare dotato di una lampada a fluorescenza e filtri di emissione adeguati. Trasferire gli embrioni positivi in ​​una piastra di Petri sterile contenente medio 1x E3. </li><li> Embrioni Dechorionate sotto uno stereomicroscopio con pinze 12. </li><li> Trasferimento un embrione in una provetta da 1,5 ml contenente 1,0 ml di pre-riscaldato 1,0% agarosio basso punto di fusione (LMA) preparato in acqua ultrapura utilizzando una pipetta di cut-tip (P200). Nota: Riscaldare la LMA a 80 ° C e raffreddare per 3-5 minuti a temperatura ambiente prima di incorporare l&#39;embrione. </li><li> Trasferire l&#39;embrione in 150 ml LMA in unchambered coprioggetti e regolare l&#39;orientamento dell&#39;embrione, ad esempio lato dorsale giù nell&#39;esperimento descritto per la invertita laser confocale microscopio a scansione A1R + (o qualsiasi altro microscopio adatto), utilizzando una punta plastica fine. Quando la LMA è polimerizzato, riempire la camera con pesci d&#39;acqua contenente 0,2 mM PTU e 0,02% di etile 3 amminobenzoato methansulfonate (Tricaine) per l&#39;anestesia. </li><li> Prima di imaging del campione, attendere 15 min per accertare l&#39;effetto di Tricaine. L&#39;anestesia impedisce i movimenti di embrioni, che possono essere monitorati utilizzando uno stereomicroscopio dotato di illuminazione campo chiaro. Nota: Le camere possono essere riutilizzati due volte. </li></ol> 4. PSmOrange Fotoconversione <ol><li> Posizionare il campione sotto il microscopio confocale e scansione dei campioni per identificare l&#39;area da photoconvert utilizzando 488 nm e 561 nm laser per l&#39;imaging rispettivamente GFP e PSmOrange. Nota: Utilizzare il invertito confocale a scansione laser microscopio A1R + sul STA invertitage TiEcontrolled dal software di imaging del microscopio e dotato di 488 nm, 561 nm e 640 nm laser per fotoconversione e di imaging. Tuttavia, l&#39;applicazione non è certamente limitato a questo microscopio finché le linee laser per la conversione e l&#39;imaging sono disponibili. <ol><li> Mettere l&#39;obiettivo di aria 20X in posizione (NA: 0,75; WD: 1,0 mm, FOV: 0,64 x 0,64 mm). </li><li> Utilizzare le seguenti impostazioni microscopio per rilevare la proteina PSmOrange prima fotoconversione: 561 nm laser, 0.74 mW misurata in corrispondenza del piano di messa a fuoco sopra l&#39;obiettivo. Nota: Questo corrisponde al 40% su questo sistema (misura nel piano focale è l&#39;unico modo per determinare la potenza effettiva). Regolare la potenza del laser in base alle esigenze sperimentali come l&#39;efficienza delle iniezioni H2B-PSmOrange può variare. </li><li> Aggiungere fattori di zoom per evidenziare l&#39;area di interesse. ingrandimenti maggiori riducono i tempi di acquisizione delle immagini e quindi fototossicità. </li></ol></li><li> Acquisire un z-stack che copre la strutturas di interesse utilizzando 488 nm e 561 nm laser in modalità sequenziale (modalità riga 1-&gt; 4). Fissare la z-passo tra 1,0 e 2,0 micron. media e la frequenza di scansione possono essere utilizzate per ottimizzare la qualità dell&#39;immagine. </li><li> Scansione campione e seleziona la regione di interesse utensile (ROI) per evidenziare la zona per photoconvert. Fissare il ROI come area di stimolazione. Selezionare il modulo Photo Attivazione / candeggio, attivare il laser 488 nm selezionando la rispettiva casella e impostare il laser 488 nm a 80% (1 mW potenza laser misurato allo scopo). Impostare la velocità di scansione di 0,5 sec / Frame. </li><li> Aprire l&#39;utilità fotoconversione (ND Stimulation) e specificare le impostazioni fotoconversione. <ol><li> Fare clic sul comando &quot;Aggiungi&quot; per specificare il protocollo fotoconversione. </li><li> Impostare &quot;Fase&quot; 1 nel menu &quot;acquisizione / stimolazione&quot; a &quot;Acquisizione&quot; e indicare il numero di immagini da acquisire prima fotoconversione nel menu &quot;Loop&quot;. </li><li> Impostare &quot;Fase&quot; 2 su &quot;Stimulation &quot;e inserire il numero di eventi di stimolazione da eseguire. </li><li> Regolare &quot;Fase&quot; 3 come descritto per &quot;Fase&quot; 1. Facoltativamente, inserire una attesa &quot;Fase&quot; dopo &quot;Fase&quot; 2 inserendo il tempo di attesa prima che l&#39;ultimo round di acquisizione delle immagini. </li><li> Una volta impostati tutti i parametri, applicare l&#39;impostazione stimolazione ed eseguire il fotoconversione. Nota: potenza del laser, la frequenza di scansione e numero di iterazioni per ogni turno di fotoconversione può variare e differiscono da embrione embrione. Un ambiente per cominciare è mostrato in Tabella 1. </li></ol></li><li> Acquisire un z-stack finale utilizzando 488 nm, 561 nm e 640 nm laser che applicano le impostazioni come sopra. Impostare il laser 640 nm per visualizzare la PSmOrange convertito con alto potere del laser (fino a 4,5 mW). </li></ol> 5. Smontare embrione da LMA <ol><li> Rimuovere l&#39;embrione camera di contenimento dal microscopio. Rimuovere con attenzione l&#39;embrione dalla u agarosiocantare pinze. </li><li> Trasferire l&#39;embrione in una nuova plastica sterilizzati petri-piatto o in un 6-pozzetti-sterilizzati contenente 1x E3 e 0,2 mM PTU. Incubare l&#39;embrione a 28 ° C fino alla fase di sviluppo desiderato. </li></ol> 6. Analizzando il destino di Photoconverted PSmOrange proteine ​​cellule che esprimono <ol><li> Re-inserire l&#39;embrione come descritto ai punti 3.3 e 3.4. </li><li> Posizionare il campione sotto il microscopio confocale e la scansione del campione per identificare le cellule photoconverted (640 nm) nella struttura transgenico GFP di interesse (488 nm). </li><li> Acquisire un z-stack che copre le strutture di interesse utilizzando 488 nm, 561 nm e 640 nm laser in modalità sequenziale (modalità riga 1-&gt; 4). Fissare la z-passo tra 1,0 e 2,0 micron. media e la frequenza di scansione possono essere utilizzate per ottimizzare la qualità dell&#39;immagine. </li><li> Utilizzare uno dei seguenti metodi per identificare le cellule, che GFP co-express e la proteina photoconverted. <ol><li> Una soluzione su misura automatica Fiji ImmagineJ 3 Macro <ol><li> Convolve lo stack originale utilizzando il plugin &quot;GaussianBlur&quot; (→ Processo → Filtri → GaussianBlur) e sottrarre le pile lisce dall&#39;originale utilizzando il plug-in &quot;Immagine calcolatore&quot; (→ Processo → calcolatore di immagine). Utilizzare questo passaggio per visualizzare le strutture di interesse e di ridurre il tempo di calcolo per l&#39;analisi. </li><li> Applicare soglie specifiche per i canali verde e lontano-rosso al fine di evidenziare le cellule photoconverted (→ Immagine → Regola → Soglia). Utilizzare lo strumento &quot;Analisi Particelle&quot; per rilevare le aree di soglia con un ambiente adatto (→ Analisi → Analisi Particelle). </li><li> Aprire il plugin &quot;immagine Calcolatore&quot; e visualizzare i ROI sovrapposte nel canale verde e lontano-rosso in giallo (→ Processo → Immagine Calculator). </li></ol></li><li> software di imaging Microscopio per la valutazione dei dati 3D <ol><li> Visualizzare lo stack in3D utilizzando l&#39;opzione di visualizzazione Mostra volume (→ Visualizzazione 3D Menu → Visualizza Volume View). Utilizzare l&#39;interfaccia grafica per selezionare i canali verde, rosso e rosso lontano, regolare la luminosità ed il contrasto e ritagliare stack 3D per evidenziare l&#39;area photoconverted (Figura 1). Nota: Metodi simili per analizzare sono disponibili i dati nella maggior parte del software comune per l&#39;analisi delle immagini. </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

embrioni transgenici portatori giornalisti fluorescenti hanno contribuito fondamentalmente a comprendere lo sviluppo embrionale. Tuttavia, vi è ancora la necessità essenziale per i promotori per facilitare la visualizzazione specifica di particolari strutture. In loro assenza, i ricercatori si basano su tecniche come la fotoconversione di proteine ​​fluorescenti per scoprire l&#39;origine e lo sviluppo della loro struttura di interesse. Questo a sua volta è un prerequisito fondamentale per identificare i meccanis...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table><tbody><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Plastic Pasteur</td><td>alpha laboratories</td><td>LW4000</td><td></td></tr><tr><td>Original H2B-PSmOrange Plasmid</td><td>Addgene</td><td>31920</td><td>The plasmid described in the paper is available in the Carl lab</td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.013</td><td></td></tr><tr><td>Forceps (5 Inox)</td><td>NeoLab</td><td>2-1633</td><td></td></tr><tr><td>Lab-Tek II Chambered #1.5 German Coverglass System&amp;nbsp;</td><td>Nunc</td><td>155382</td><td></td></tr><tr><td>Nikon A1R+</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>Nikon PLAN Apo &amp;lambda; 20X air objective&amp;nbsp;</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>NIS Elements AR Software (v. 4.30.02)</td><td>Nikon GmbH Germany/Laboratory Imaging</td><td>No Number</td><td></td></tr></tbody></table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

La Figura 1 illustra un esempio del sistema PSmOrange fotoconversione. Il complesso pineale è una struttura conservata nel diencefalo dorsali vertebrati. Come in molti altri vertebrati, il complesso costituito dall&#39;organo pineale nel centro del diencefalo e le cellule parapineal sinistre. Elegante ma in termini di tempo esperimenti uncaging hanno dimostrato che le cellule parapineal hanno origine nella parte anteriore dell&#39;organo pineale 13. In tg (fo...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Monitoraggio celle in Zebrafish GFP-transgenico Utilizzo del sistema fotoconvertibile PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.5K Views.  Heidelberg University. We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Video: Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Cella singola fotoconversione in Zebrafish intatto vivente

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Ricerca

Biologia dello sviluppo

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Seguendo i movimenti del tessuto Endocardial tramite cella fotoconversione nell'embrione di pesce zebra

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

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

Biologia

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Fotoconversione di proteine ​​purificate fluorescenti ed evidenziazione ottica dual-sonda in cellule vive

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor time-lapse imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Singolo Destino Mapping di celle in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Cella di rilevamento utilizzando proteine ​​Photoconvertible durante lo sviluppo Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Generazione di Dispersi Presomitic mesoderma colture cellulari, per Immagini del Zebrafish segmentazione Orologio in celle singole

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

Imaging cellulare vivo ROS durante lo sviluppo neuronale

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Neuroscienze

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

Saggio di separazione di fase proteica: un metodo optogenetico per la separazione di fase proteica legante l'RNA mutante nei motoneuroni spinali di larve di zebrafish

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...

Monitoraggio celle in Zebrafish GFP-transgenico Utilizzo del sistema fotoconvertibile PSmOrange

Riepilogo

Esplora Altri Video

Capitoli in questo video

Cella singola fotoconversione in Zebrafish intatto vivente

Seguendo i movimenti del tessuto Endocardial tramite cella fotoconversione nell'embrione di pesce zebra

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

Fotoconversione di proteine purificate fluorescenti ed evidenziazione ottica dual-sonda in cellule vive

Multicolor time-lapse imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

Singolo Destino Mapping di celle in Zebrafish

Cella di rilevamento utilizzando proteine Photoconvertible durante lo sviluppo Zebrafish

Generazione di Dispersi Presomitic mesoderma colture cellulari, per Immagini del Zebrafish segmentazione Orologio in celle singole

Imaging cellulare vivo ROS durante lo sviluppo neuronale

Saggio di separazione di fase proteica: un metodo optogenetico per la separazione di fase proteica legante l'RNA mutante nei motoneuroni spinali di larve di zebrafish

Monitoraggio celle in Zebrafish GFP-transgenico Utilizzo del sistema fotoconvertibile PSmOrange

Riepilogo

Esplora Altri Video

Capitoli in questo video

Cella singola fotoconversione in Zebrafish intatto vivente

Seguendo i movimenti del tessuto Endocardial tramite cella fotoconversione nell'embrione di pesce zebra

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

Fotoconversione di proteine ​​purificate fluorescenti ed evidenziazione ottica dual-sonda in cellule vive

Multicolor time-lapse imaging di transgenici Zebrafish: Visualizzare cellule staminali della retina Attivato da mirata ablazione delle cellule neuronali

Singolo Destino Mapping di celle in Zebrafish

Cella di rilevamento utilizzando proteine ​​Photoconvertible durante lo sviluppo Zebrafish

Generazione di Dispersi Presomitic mesoderma colture cellulari, per Immagini del Zebrafish segmentazione Orologio in celle singole

Imaging cellulare vivo ROS durante lo sviluppo neuronale

Saggio di separazione di fase proteica: un metodo optogenetico per la separazione di fase proteica legante l'RNA mutante nei motoneuroni spinali di larve di zebrafish

Fotoconversione di proteine purificate fluorescenti ed evidenziazione ottica dual-sonda in cellule vive

Cella di rilevamento utilizzando proteine Photoconvertible durante lo sviluppo Zebrafish