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.

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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 border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Materials:</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>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>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pair of Dumont #5 forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cover-glass-bottomed culture dish</td>
 <td>MatTek Corp</td>
 <td>&nbsp;</td>
 <td>Ashland MA USA</td>
 </tr>
 <tr>
 <td>Microloader Pipette Tips</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
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 <td>Vortex</td>
 <td>&nbsp;</td>
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 <td>needles</td>
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 <td>&nbsp;</td>
 <td>Reagents:</td>
 </tr>
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 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Egg water medium</td>
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 <td>60 &mu;g/ml Instant Ocean Sea Salts in ddH2O</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
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 <td>E3 medium </td>
 </tr>
 <tr>
 <td>5 mM NaCl</td>
 <td>&nbsp;</td>
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 <td>0.17 mM KCl</td>
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 <td>0.33 mM CaCl2</td>
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 <td>0.33 mM MgSO4</td>
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 <td>0.00001% (w/v) Methylene Blue</td>
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 <td>4 mg/ml Tricaine stock solution </td>
 </tr>
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 <td>400 mg Tricaine (3-amino benzoic acidethylester)</td>
 <td>Sigma-Aldrich</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
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 <td>97.9 ml ddH2O</td>
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 <td>2.1 ml 1M Tris (pH9)</td>
 <td>&nbsp;</td>
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 <td>Adjust at pH 7, aliquot and store -20 &deg;C</td>
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 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1% LMA (Low melting temperature agarose, Lonza)</td>
 </tr>
 <tr>
 <td>1g LMA/100 ml E3 medium 1X</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aliquot and store at 4 &deg;C</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</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.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

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

Research

JoVE Journal

Biology

11.3K ビュー.  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.

ビデオ: Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

CyDye DIGEフルーア最小限の色素を用いた細胞表面タンパク質の選択的ラベリング

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

生物学

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

ショウジョウバエのフライトの間に視覚的に仲介臭気の追跡

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

セプターのセルカウンターを用いて細胞培養中の細胞数の決定

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

ガス透過性材料技術とHYPERStackの船舶を使用するクローズドシステムの細胞培養のプロトコル

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

胚発生時に細胞運命のコミットメントをテストするために割球植

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D共焦点顕微鏡を用いたゼブラフィッシュにおける顔面形態形成を分析する

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

蛍光クリアランスアッセイを用いてゼブラフィッシュ腎臓機能の評価

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...