This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

All mice were housed and handled in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and enrolled in an NHLBI Animal Care and Use Committee&#8211;approved protocol. Female B6.SJL-Ptprc(d)Pep3(b)/BoyJ (B6.SJL) and C57Bl/6 mice, 6-12 weeks old, were used as donor and recipient, respectively.
A list of materials, reagents, and equipment is provided in Table 1.
1. LeGO Transduction of Mouse HSPCs a...

<ol>
	<li>Lo Celso, ., C, D. T., Scadden, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124, 3529-3535 (2011).</li><li>Lo Celso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Takaku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis+in+3+dimensions+human+and+murine+bone+marrow+architecture+visualized+by+confocal+microscopy.">Hematopoiesis in 3 dimensions human and murine bone marrow architecture visualized by confocal microscopy.</a> Blood. 116, e41-e55 (2010).</li><li>Malide, D., Metais, J. Y., Dunbar, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clonal+analysis+of+murine+hematopoietic+stem+and+progenitor+cells+marked+by+5+fluorescent+proteins+using+confocal+and+multiphoton+microscopy.">Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.</a> Blood. 120, e105-e116 (2012).</li><li>Weber, K., Mock, U., Petrowitz, B., Bartsch, U., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+ontology+(LeGO)+vectors+equipped+with+novel+drug-selectable+fluorescent+proteins+new+building+blocks+for+cell+marking+and+multi-gene+analysis.">Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins new building blocks for cell marking and multi-gene analysis.</a> Gene Ther. 17, 511-520 (2010).</li><li>Weber, K., Bartsch, U., Stocking, C., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolor+panel+of+novel+lentiviral+'gene+ontology'+(LeGO)+vectors+for+functional+gene+analysis.">A multicolor panel of novel lentiviral 'gene ontology' (LeGO) vectors for functional gene analysis.</a> Mol Ther. 16, 698-706 (2008).</li><li>Weber, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGB+marking+facilitates+multicolor+clonal+cell+tracking.">RGB marking facilitates multicolor clonal cell tracking.</a> Nat Med. 17, 504-509 (2011).</li><li>Lo Celso, ., Lin, C. 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The authors declare no competing financial interests.

We describe here the details of a powerful methodology recently devised for clonal cell tracking, combining the large diversity generated by combinatorial marking with 5FP-encoding LeGO vectors and imaging with tandem confocal and 2-photon microscopy to achieve volumetric and dynamic imaging in live tissues. This extended the use of FP-based color marking by recording confocal spectral identity in 5 &#8220;distinct&#8221; 8-bits channels. Thus the relative ratio of Cerulean, EGFP, Venus, tdTomato, mCherry within each cel...

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

The production of blood cells, termed hematopoiesis, is maintained by a small population of hematopoietic stem and progenitor cells (HSPCs). These cells reside within the bone marrow (BM) in a complex microenvironmental niche consisting of osteoblasts, stromal cells, adipose tissue, and vascular structures, all implicated in the control of self-renewal and differentiation1,2. As intact BM has been traditionally inaccessible to direct observations, the interactions between HSPC and their microenvironment remains largely uncharacterized in vivo. Previously, we established a methodology to visualize the 3D architecture of intact BM using confocal fluorescence and reflection microscopy3. We characterized expansion of EGFP-marked HSPCs in the BM, but the use of only a single FP precluded analysis of regeneration at a clonal level. Very recently we took advantage of the Lentiviral Gene Ontology (LeGO) vectors constitutively expressing fluorescent proteins (FPs) to efficiently mark cells4,5. Co-transduction of HSPC with 3 or 5 vectors generates a diverse palette of combinatorial colors, allowing tracking of multiple individual HSPC clones.
Marked HSPC combined with new imaging technology permitted to trace individual HSPC homing and engraftment in the BM of irradiated mice. LeGO vectors were used to mark HSPC with 3 or 5 different FPs (from Cerulean, eGFP, Venus, tdTomato, and mCherry) and follow their engraftment over time in the BM by confocal and 2-photon microscopy, allowing clear visualization of bone and other matrix structures, and the relation of HSPC clones to components of their microenvironment. HSPC were purified as the lineage negative cells from C57Bl/6 mice BM, transduced with LeGO vectors, and reinfused by tail vein injection into myelo-ablated congenic mice. By monitoring large volumes of the sternum BM tissue we visualized endosteal engraftment occurring early in BM regeneration. Cell clusters with diverse color spectra appeared initially in close proximity to the bone and progressed centrally over time. Interestingly, after more than 3 weeks the marrow consisted of macroscopic clonal clusters, suggesting spread of hematopoiesis derived from individual HSPCs contiguously in the marrow, rather than widespread dissemination of HSPCs via the bloodstream. There was less color diversity at late time points, suggesting difficulty in transducing long-term repopulating cells with multiple vectors.
In addition, HSPCs formed clusters in the spleen, an organ also responsible for hematopoiesis in mice. HPSC-derived individual cells could be resolved in the thymus, lymph nodes, spleen, liver, lung, heart, skin, skeletal muscle, adipose tissue, and kidney as well. The 3D images can be assessed qualitatively and quantitatively to appreciate the distribution of cells with minimal perturbations of the tissues. Finally, we illustrated the feasibility of live dynamic studies in 4D by combining resonant scanning multiphoton and confocal time-lapse imaging. This methodology enables non-invasive high resolution, multidimensional cell-fate tracing of spectrally marked cells populations in their intact 3D architecture, providing a powerful tool in the study of tissue regeneration and pathology.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Production of viruses</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-Cer2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27338</td>
			<td>Expression of Cerulean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-G2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">25917</td>
			<td>Expression of eGFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-V2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27340</td>
			<td>Expression of Venus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-T2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27342</td>
			<td>Expression of tdTomato</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-C2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27339</td>
			<td>Expression of mCherry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Calciumm Phosphate Transfection Kit</td>
			<td>Sigma</td>
			<td>CAPHOS-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Tissue culture dish</td>
			<td style="width:493px;">BD Falcon</td>
			<td style="width:216px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">IMDM</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Fetal Bovine Serum Heat Inactivated</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pen Strep Glutamine</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Centrifuge Tubes</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">326823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SW28 Ultracentrifuge</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">L60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Millex Syringe Driven Filter Unite (0.22um)</td>
			<td style="width:493px;">Millipore</td>
			<td style="width:216px;">SLGS033SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse cell collection, purification, transduction and transplantation</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">ACK lysing buffer</td>
			<td style="width:493px;">Quality Biologicals Inc.</td>
			<td style="width:216px;">118-156-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Lineage Cell Depletion Kit (mouse)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LS columns + tubes</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pre-Separation Filters (30um)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:493px;">StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500mL)</td>
			<td>StemCell Technologies Inc&#160;</td>
			<td>9650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">murine IL-11 CF</td>
			<td>R &#38; D Systems Inc</td>
			<td>418-ML-025/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine SCF</td>
			<td style="width:493px;">RDI Division of Fitzgerald Industries Intl&#160;</td>
			<td>RDI-2503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine IL-3</td>
			<td>R &#38; D Systems Inc</td>
			<td>403-ML-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">FLT-3 Ligand</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-096-480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">12-well plates, Costar</td>
			<td style="width:493px;">Corning Inc.</td>
			<td style="width:216px;">3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Retronectin</td>
			<td style="width:493px;">Takara Bio Inc</td>
			<td style="width:216px;">T100A/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Protamine Sulfate</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">P-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Confocal and two-photon microscopy</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">DMEM</td>
			<td style="width:493px;">Lonza</td>
			<td style="width:216px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">1M Hepes</td>
			<td style="width:493px;">Cellgro, Mediatech Inc.</td>
			<td style="width:216px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-20-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-14-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td>
			<td>Thermo Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Vision II -TiSaph laser range 680-1080nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Compact OPO laser range 1030-1350nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCX-IRAPO-L 25x/0.95 NA water dipping objective (WD=2.5 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PLAPO-CS 20x/0.70 NA dry objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PL-IRAPO 40x/1.1NA water immersion objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Imaris software version 7.6</td>
			<td style="width:493px;">Bitplane</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

Whole-mount 3D confocal/2-photon microscopy reconstructions of the sternal bone marrow time course revealed the engraftment and expansion of transplanted co-5FPs in a pattern with remarkable characteristics: clones appeared clearly delineated, homogenously marked with wide palette of colors initially and progress over time to preferentially contain cells of mostly one color. Confocal microscopy setup and representative examples of imaging 5FPs-marked HSPC in the sternal bone marrow are illustrated in Figure 2</st...

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

in-vivo-clonal-tracking-hematopoietic-stem-progenitor-cells-marked

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JoVE Journal

Biology

13.1K Views.  NHLBI/NIH. Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation. 

Video: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy