We thank Dr. Ian C. Scott for kindly providing the zebrafish transgenic Tg(kdrl:nlsKikGR)hsc7 line at the Hospital for Sick Children, Toronto, Canada. We thank the Confocal and 2-Photon Microscopy Core Facility (Max-Delbrueck-Center for Molecular Medicine) and Dr. Zoltan Cseresnyes, for excellent technical assistance and overall imaging support. S.A.-S. is supported by a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

1. Obtaining Embryos for the Photoconversion Assay
Upon in-crossing of transgenic zebrafish expressing a PCFP the brightest which are homozygote embryos are chosen, and grown at 28.5 &deg;C until the desired embryonic stage15.
Note 1: This preselection will allow scanning embryos with low laser power thereby preventing phototoxicity and photobleaching.
Note 2: We suggest to use low-intensity light when visualizing embryos and to maintain them in the dark conditions due to high sensitivity of PCFPs, such as Kaede and KikGR, to light exposure including daylight.
2. Embedding
<ol>
	<li>In a glass container with egg-water, dechorionate embryos under stereomicroscope using forceps.</li>
	<li>Anesthetize embryos by transferring a dechorionated embryo into 0.16 mg/ml Tricaine (3-amino benzoic acidethylester, Sigma-Aldrich) solution (40&mu;l of 4 mg/ml stock solution in 1 ml of E3 medium 1X).</li>
</ol>
Note 1: Transfer embryos with a glass pipette or a cut-tip (P1000) to minimize the risk of damaging the embryos.
Note 2: Young dechorionated embryos should not be exposed to air or lifted up to the water/air interface, since the eggs easily rupture when subjected to mechanical stress.
<ol start="3">
	<li>Transfer the anesthetized embryo onto the cover of a culture dish and maintain the embryo within the smallest possible volume of medium.</li>
	<li>Transfer the embryo into agarose by pipetting the embryo with 1% low melting temperature agarose (LMA, Lonza) at 30 &deg;C containing 0.16 mg/ml Tricaine, and place it onto the cover-glass of a cover-glass-bottomed culture dish (MatTek Corp. Ashland MA USA). Subsequently, orient the embryo with the help of a smooth plastic tip (e.g. a cut Microloader Pipette Tip, Eppendorf) and, when the LMA has polymerized, cover agarose with 0.16 mg/ml Tricaine solution.</li>
</ol>
Note 1: Melt 1% LMA at 60 &deg;C and maintain melted agarose at 30 &deg;C.
3. Photoconversion
<ol>
	<li>On an inverted confocal microscope equipped with an 405 nm, 488nm and 561nm laser source, such as the Zeiss LSM 710, visualize the sample by a z-Stack scan covering the entire structure of the specimen to be photoconverted and tracked later on using the 488 nm laser (i.e., 30 mW Ar laser,at 5-7 % intensity, detector set at 493-540 nm) and the 561 nm laser (i.e., 10 mW DPSS 561 laser, at 7-9 % intensity, detector set at 587-651) to scan the tissue/organ for the green and red fluorescence of the photoconvertible proteins, respectively.</li>
</ol>
Note 1: For the scan before photoconversion, we used a Plan Apochromat 20x/NA 0.8 objective.
Note 2: Always scan for the red fluorescence prior to the experiment to exclude that any accidentally generated red converted PCFP is present.
<ol start="2">
	<li>Select the Time series tool and set for 2 cycles with no interval (one for pre- and one for post photoconvertion).</li>
	<li>Select the Regions tool and define one or more regions of interest (ROI) for photoconversion.</li>
</ol>
Note 1: We recommend to first determine the precision with which the region actually photo-converted by the scanning laser in the ROI matches the ROI selected in the software by measuring the area of photoconverted tissue/cells in all three (xy and z direction) in a fixed specimen where no cell or protein movement is expected. For the settings described within this application, the ROI matched the photoconverted region with great precision but this depends on the objective used and the amount of laser applied for photoconversion. The size of the ROI can be reduced to the size of a single cell depending on the numerical aperture of the objective, the zoom factor and the amount of laser light used for photoconversion.
<ol start="4">
	<li>Select the Bleaching tool and set for start bleaching after scan 1 of 2. We used a 30 mW 405 nm diode laser for photoconversion and carefully optimized for the minimal amount of laser light necessary for complete photoconversion which is mostly achieved by varying the intensity of the laser light, the scan iterations of the ROI and the pixel time (i.e., scan speed).</li>
</ol>
Note 1: For photoconversion we used a Plan Apochromat 20x/NA 0.8 objective.
Note 2: The power of the laser and the number of iterations must be determined empirically for each experimental setting and should ensure the complete photoconversion from green to red PCFP within the ROI with the minimal necessary amount of laser light to reduce phototoxicity. We recommend to initiate tests using low laser power (e.g. 3 %, corresponding to 0.03 mW in the objective plan) combined with low numbers of iterations (e.g. 20), and to subsequently increase the laser power until complete photoconversion is achieved which in our hands was at 10% laser power (corresponding to 0.1 mW in the objective plan). Under these conditions, all embryos tested survived the treatment plan. Nevertheless, we observed that the yolk membrane is sensitive to laser pulses and can easily be damaged causing death of the embryo. For this reason, embryo lethality after photoconversion may be caused if the position of the target tissue is close to the yolk body.
<ol start="5">
	<li>Scan the sample by a z-Stack using lasers for 488 nm and 561 nm to visualize any remaining green and the photoconverted red fluorescence of the PCFP.</li>
</ol>
Note 1: For the scan after photoconversion, we used a Plan Apochromat 20x/NA 0.8.
4. Remove Embryo from Agarose
<ol>
	<li>Discard the solution and carefully remove the embryo from the 1% LMA with the help of a needle or a smooth plastic tip.</li>
	<li>Maintain the embryo in egg water in the dark at 28.5 &deg;C until the desired developmental stage.</li>
</ol>
Note 1: It is important that embryos continue their development in egg water, particularly during the first days of development during extension of the embryonic body. Indeed, some embryonic tissues/organs change positions during development and hence require specific orientations during embedding which depends on the particular tissue/organ of interest and the desired developmental stage of analysis.
5. Re-embedding
<ol>
	<li>Proceed like under point 2.) of this protocol.</li>
</ol>
6. Tracking Photoconverted Tissue at Later Embryonic Stages
<ol>
	<li>Scan of the region of interest (z-Stack) using lasers for 488 nm and 561 nm for green and red fluorescent signals, respectively.</li>
</ol>
Note 1: For the scan x hours post-photoconversion, we used a Plan Apochromat 20x/NA 0.8.
Note 2: As this protocol does not affect the normal development of the embryo, it is possible to observe the photoconverted embryo at later stages of development (for this, proceed as outlined under points 4 - 6).
7. Representative Results
An example of a photoconversion assay is shown in Figure 1. We tracked endothelial cells during zebrafish development (for a review of the vascular anatomy of the developing zebrafish see16). Accordingly, we used a zebrafish Tg(kdrl:nlsKikGR)hsc7 reporter line expressing KikGR within endothelial nuclei17. Homozygous and brightly expressing embryos were raised at 28.5 &deg;C until 48 hr post fertilization (hpf). Upon dechorionation, embryos were anesthetized and embedded in LMA positioning the anterior-dorsal side of the embryo towards the cover glass for better visualization of head vessels. We use an inverted confocal microscope (Zeiss LSM 710 Meta) equipped with lasers operating at 405 nm, 488 nm and 561 nm, and a Plan Apochromat 20x/NA 0.8 objective for the photoconversion assay and further tracking of the photoconverted protein. As shown in Figure 1A, before photoconversion, KikGR was expressed within endothelial tissue and was only detectable as green fluorescence using a 488 nm Argon laser. Control scans with a 561 nm DPSS 561-10 laser for red fluorescence did not detect any photoconverted KikGR prior to the photoconversion (Figure 1A). Subsequently, the ROI was completely photoconverted using a 405 nm Diode laser (at 10% intensity, 20 iterations) and the head vessels were scanned again using the 488 nm and 561 nm lasers (Figure 1B). As shown in Figure 1B, green KikGR had completely switched to red within the ROI. After the photoconversion assay, the embryo was removed from the agarose and raised at 28.5 &deg;C in egg water. Finally, the embryo was observed again 24 hr post-photoconversion, around 72 hpf, to track the photoconverted endothelial cells at later stages (Figure 1C). Thus, the embryo was embedded in LMA, and the sample was re-scanned using the 488 nm and 561 nm lasers. As shown in Figure 1C, within endothelial tissue arising from the photoconverted ROI, both non- and photoconverted KikGR proteins were observed due to the stability of the red fluorescent photoconverted KikGR and re-synthesis of new green non-photoconverted KikGR.
<img alt="Figure 1" src="/files/ftp_upload/4350/4350fig1.jpg" /> 
Figure 1. Photoconversion of KikGR during zebrafish development. (A,B) A specific region of the endothelial head vasculature was photoconverted at 48 hpf in Tg(kdrl:nlsKikGR)hsc7 transgenic embryos and (C) observed again 24 hr post-photoconversion (around 72 hpf). The fluorescence of KikGR (green), photoconverted KikGR (red) and merged channels are shown. Dotted circles show the ROI before, after, and 24 hr post- photoconversion. Images are dorsal views of the zebrafish embryo, anterior to the bottom. Scale bars: 50 &mu;m.
<table border="1">
	<tbody>
		<tr>
			<td>Protein (Acronym)</td>
			<td>Ex (nm)</td>
			<td>Em (nm)</td>
			<td>EC </td>
			<td>QY</td>
			<td>Quaternary Structure</td>
			<td>Brightness </td>
			<td>Application</td>
			<td>Ref</td>
		</tr>
		<tr>
			<td>Kaede_G</td>
			<td>508</td>
			<td>518</td>
			<td>99</td>
			<td>0.9</td>
			<td>tetramer</td>
			<td>259</td>
			<td rowspan="2">Cell tracking</td>
			<td rowspan="2">2,6</td>
		</tr>
		<tr>
			<td>Kaede_R</td>
			<td>572</td>
			<td>580</td>
			<td>60</td>
			<td>0.3</td>
			<td>tetramer</td>
			<td>59</td>
		</tr>
		<tr>
			<td>KikGR_G</td>
			<td>507</td>
			<td>517</td>
			<td>54</td>
			<td>0.7</td>
			<td>tetramer</td>
			<td>112</td>
			<td rowspan="2">Cell tracking</td>
			<td rowspan="2">3,6,8-10 </td>
		</tr>
		<tr>
			<td>KikGR_R</td>
			<td>583</td>
			<td>593</td>
			<td>35</td>
			<td>0.6</td>
			<td>tetramer</td>
			<td>68</td>
		</tr>
		<tr>
			<td>mKikGR_G</td>
			<td>505</td>
			<td>515</td>
			<td>49</td>
			<td>0.7</td>
			<td>monomer</td>
			<td>101</td>
			<td rowspan="2">Dynamic tracking Superresolution images</td>
			<td rowspan="2">13</td>
		</tr>
		<tr>
			<td>mKikGR_R</td>
			<td>580</td>
			<td>591</td>
			<td>28</td>
			<td>0.6</td>
			<td>monomer</td>
			<td>53</td>
		</tr>
		<tr>
			<td>EosFP_G</td>
			<td>506</td>
			<td>516</td>
			<td>72</td>
			<td>0.7</td>
			<td>tetramer</td>
			<td>150</td>
			<td rowspan="2">Cell tracking</td>
			<td rowspan="2">5,20</td>
		</tr>
		<tr>
			<td>EosFP_R</td>
			<td>571</td>
			<td>581</td>
			<td>41</td>
			<td>0.5</td>
			<td>tetramer</td>
			<td>67</td>
		</tr>
		<tr>
			<td>tdEos_G</td>
			<td>506</td>
			<td>516</td>
			<td>34</td>
			<td>0.7</td>
			<td>tandem dimer</td>
			<td>165</td>
			<td rowspan="2">Superresolution images</td>
			<td rowspan="2">12,14,21</td>
		</tr>
		<tr>
			<td>tdEos_R</td>
			<td>569</td>
			<td>581</td>
			<td>33</td>
			<td>0.6</td>
			<td>tandem dimer</td>
			<td>59</td>
		</tr>
		<tr>
			<td>mEos2_G</td>
			<td>506</td>
			<td>519</td>
			<td>56</td>
			<td>0.7</td>
			<td>monomer</td>
			<td>140</td>
			<td rowspan="2">Dynamic tracking Superresolution images</td>
			<td rowspan="2">11,18</td>
		</tr>
		<tr>
			<td>mEos2_R</td>
			<td>573</td>
			<td>584</td>
			<td>46</td>
			<td>0.7</td>
			<td>monomer</td>
			<td>90</td>
		</tr>
		<tr>
			<td>Dendra2_G</td>
			<td>490</td>
			<td>507</td>
			<td>45</td>
			<td>0.5</td>
			<td>monomer</td>
			<td>67</td>
			<td rowspan="2">Dynamic tracking Superresolution images</td>
			<td rowspan="2">4,11</td>
		</tr>
		<tr>
			<td>Dendra2_R</td>
			<td>553</td>
			<td>573</td>
			<td>35</td>
			<td>0.55</td>
			<td>monomer</td>
			<td>57</td>
		</tr>
	</tbody>
</table>
Table I. Properties of photoconvertable fluorescent proteins. Along with the common name and/or acronym for each PCFPs, the peak excitation (Ex) and emission (Em) wavelengths, molar extinction coefficients (EC, 10-3 M-1 cm-1 ), quantum yield (QY), physiologically relevant quaternary structure, relative brightness (as % of EGFP), application, and references are shown in the table. Table adapted from Day and Davidson, 20101.
<table border="1">
	<tbody>
		<tr>
			<td>PCFP</td>
			<td>Zebrafish transgenic lines</td>
			<td>Source</td>
		</tr>
		<tr>
			<td rowspan="10">Kaede</td>
			<td>Tg(elavl3:Kaede)rw0130a</td>
			<td>Okamoto Lab</td>
		</tr>
		<tr>
			<td>Tg(EPV.TP1-Mmu.Hbb:Kaede)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(ins:Kaede)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(isl1:Gal4-VP16,14xUAS:Kaede)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(lhx5:Kaede)b1204/+</td>
			<td>ZIRC</td>
		</tr>
		<tr>
			<td>Tg(myl7:kaede)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(olig2:Kaede)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(UAS-E1b:Kaede)s1999t</td>
			<td>ZIRC</td>
		</tr>
		<tr>
			<td>Tg(UAS:Kaede)rk8</td>
			<td>ZIRC</td>
		</tr>
		<tr>
			<td>Tg(vsx2:Kaede)nns2</td>
			<td>Higashijima Lab</td>
		</tr>
		<tr>
			<td rowspan="5">KikGR</td>
			<td>Tg(kdrl:nlsKikGR)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(krt4:NTR-KikGR)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(myl7:nlsKikGR)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(sox10:KikGR)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(UAS:KikGR)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td rowspan="2">EosFP</td>
			<td>Tg(sox10:EosFP)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(sox10:nlsEosFP)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td rowspan="2">Dendra</td>
			<td>Tg(-8mpx:Dendra2)</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tg(UAS:Dendra-kras)</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table II. Zebrafish transgenic lines expressing PCFPs. Data obtained in the Zebrafish Model Organism Database (ZFIN, <a href="http://zfin.org/" target="_blank">http://zfin.org/</a>). Availability of lines in the Zebrafish International Resource Center (ZIRC, <a href="http://zebrafish.org/zirc/" target="_blank">http://zebrafish.org/zirc/</a>) is indicated.

<ol>
	<li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chem. Soc. Rev. 38, 2887-2921 (2009).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 12651-12656 (2002).</li><li>Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-rational+engineering+of+a+coral+fluorescent+protein+into+an+efficient+highlighter.">Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.</a> EMBO Rep. 6, 233-238 (2005).</li><li>Gurskaya, N. G., Verkhusha, V. V., Shcheglov, A. S., Staroverov, D. B., Chepurnykh, T. V., Fradkov, A. F., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+monomeric+green-to-red+photoactivatable+fluorescent+protein+induced+by+blue+light.">Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.</a> Nat. Biotechnol. 24, 461-465 (2006).</li><li>Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K. D., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 15905-15910 (2004).</li><li>Hatta, K., Tsujii, H., Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+a+photoconvertible+fluorescent+protein.">Cell tracking using a photoconvertible fluorescent protein.</a> Nat. Protoc. 1, 960-967 (2006).</li><li>Stark, D. A., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+comparison+of+photoactivatable+fluorescent+proteins+in+an+avian+embryo+model.">An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.</a> Dev. Dyn. 236, 1583-1594 (2007).</li><li>Kulesa, P. M., Teddy, J. M., Stark, D. A., Smith, S. E., McLennan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+invasion+is+a+spatially-ordered+progression+into+the+head+with+higher+cell+proliferation+at+the+migratory+front+as+revealed+by+the+photoactivatable+protein,+KikGR.">Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR.</a> Dev. Biol. 316, 275-287 (2008).</li><li>Nowotschin, S., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+KikGR+a+photoconvertible+green-to-red+fluorescent+protein+for+cell+labeling+and+lineage+analysis+in+ES+cells+and+mouse+embryos.">Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos.</a> BMC Dev. Biol. 9, 49 (2009).</li><li>Griswold, S. L., Sajja, K. C., Jang, C. W., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+iUBC-KikGR+photoconvertible+transgenic+mice+for+live+time-lapse+imaging+during+development.">Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development.</a> Genesis. 49, 591-598 (2011).</li><li>Baker, S. M., Buckheit, R. W., Falk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green-to-red+photoconvertible+fluorescent+proteins:+tracking+cell+and+protein+dynamics+on+standard+wide-field+mercury+arc-based+microscopes.">Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes.</a> BMC Cell Biol. 11, 15 (2010).</li><li>Watanabe, S., Punge, A., Hollopeter, G., Willig, K. I., Hobson, R. J., Davis, M. W., Hell, S. W., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+localization+in+electron+micrographs+using+fluorescence+nanoscopy.">Protein localization in electron micrographs using fluorescence nanoscopy.</a> Nat. Methods. 8, 80-84 (2011).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKikGR,+a+monomeric+photoswitchable+fluorescent+protein.">mKikGR, a monomeric photoswitchable fluorescent protein.</a> PLoS One. 3, e3944 (2008).</li><li>Fang, Z., Takizawa, N., Wilson, K. A., Smith, T. C., Delprato, A., Davidson, M. W., Lambright, D. G., Luna, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-associated+protein,+supervillin,+accelerates+F-actin-dependent+rapid+integrin+recycling+and+cell+motility.">The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility.</a> Traffic. 11, 782-799 (2010).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Isogai, S., Horiguchi, M., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+anatomy+of+the+developing+zebrafish:+an+atlas+of+embryonic+and+early+larval+development.">The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.</a> Dev. Biol. 230, 278-301 (2001).</li><li>Lazic, S., Scott, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mef2cb+regulates+late+myocardial+cell+addition+from+a+second+heart+field-like+population+of+progenitors+in+zebrafish.">Mef2cb regulates late myocardial cell addition from a second heart field-like population of progenitors in zebrafish.</a> Dev. Biol. 354, 123-133 (2011).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nat. Methods. 6, 131-133 (2009).</li><li>Godinho, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+zebrafish+development.">Live imaging of zebrafish development.</a> Cold Spring Harb Protoc. 2011, 770-777 (2011).</li><li>Curran, K., Lister, J. A., Kunkel, G. R., Prendergast, A., Parichy, D. M., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Foxd3+and+Mitf+regulates+cell+fate+plasticity+in+the+zebrafish+neural+crest.">Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.</a> Dev. Biol. 344, 107-118 (2010).</li><li>Nienhaus, G. U., Nienhaus, K., Holzle, A., Ivanchenko, S., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Rocker, C., Vallone, B., Weidemann, W., Heilker, R., Nar, H., Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconvertible+fluorescent+protein+EosFP:+biophysical+properties+and+cell+biology+applications.">Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications.</a> Photochem. Photobiol. 82, 351-358 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li></ol>

No conflicts of interest declared.

In recent years, transgenic animal models for the expression of photoconvertible proteins such as Kaede or KikGR have been generated. These animals develop normally, indicating that these proteins have no toxic effects on embryonic development. The first report about a photoconversion assay for tracking cells in animal embryos was performed by Hatta and collaborators by injection of mRNA or DNA encoding Kaede into one-cell zebrafish embryos for ubiquitous expression, or by expression of Kaede and KikGR in neural tissue b...

<table><tbody><tr><td colspan="4">&lt;strong&gt;Materials:&lt;/strong&gt;</td></tr><tr><td colspan="4">Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).</td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>Lecia MZ6 Stereomicroscope</td><td>Equipped with laser operating at 488 nm</td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss</td><td>Zeiss LSM 710 NLO</td><td>equipped with lasers operating at 488 nm, 561 nm, and 405 nm</td></tr><tr><td>Heating block</td><td></td><td></td><td></td></tr><tr><td>Pair of Dumont #5 forceps</td><td></td><td></td><td></td></tr><tr><td>Cover-glass-bottomed culture dish</td><td>MatTek Corp</td><td></td><td>Ashland MA USA</td></tr><tr><td>Microloader Pipette Tips</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>needles</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Reagents:&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;em&gt;Egg water medium&lt;/em&gt;</td></tr><tr><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;em&gt;E3 medium &lt;/em&gt;</td></tr><tr><td>5 mM NaCl</td><td></td><td></td><td></td></tr><tr><td>0.17 mM KCl</td><td></td><td></td><td></td></tr><tr><td>0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>0.00001% (w/v) Methylene Blue</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;em&gt;4 mg/ml Tricaine stock solution &lt;/em&gt;</td></tr><tr><td>400 mg Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>97.9 ml ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>2.1 ml 1M Tris (pH9)</td><td></td><td></td><td></td></tr><tr><td>Adjust at pH 7, aliquot and store -20 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;em&gt;1% LMA (Low melting temperature agarose, Lonza)&lt;/em&gt;</td></tr><tr><td>1g LMA/100 ml E3 medium 1X</td><td></td><td></td><td></td></tr><tr><td>Aliquot and store at 4 &amp;deg;C</td><td></td><td></td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Cell Tracking Using Photoconvertible Proteins During Zebrafish Development at JoVE.com

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

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.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento 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 ...

cell-tracking-using-photoconvertible-proteins-during-zebrafish

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.3K visualizações.  Max Delbrück Center for Molecular Medicine. 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.

Vídeo: Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Medir a estabilidade da proteína em Viver embriões Zebrafish Usando Fluorescência Decay Após Fotoconversão (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Biologia do Desenvolvimento

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.

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.

Acompanhando as células GFP em peixes-zebra transgénicos Usando o Sistema Photoconvertible PSmOrange

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

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Fóton multi tempo lapso imagem para visualizar o desenvolvimento em tempo real: visualização de migrar as células da crista Neural em embriões de peixe-zebra

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

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.

Photoconversion de célula única no Zebrafish intacta vida

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 ...

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.

Seguindo os movimentos de tecido endocárdico através de célula Photoconversion no embrião Zebrafish

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

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imagens ao vivo da motilidade celular e citoesqueleto de actina de neurônios individuais e células da crista neural em embriões Zebrafish

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

Biologia

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.

Two-Photon-Based fotoativação no Live Embriões Zebrafish

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

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Um dispositivo de transplante simples e eficaz para embriões de zebrafish

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

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.

Mapeamento Destino única célula em Zebrafish

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

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Imagem 4-Dimensional da Morfogense da Copa Óptica de Zebrafish

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...