This work was supported by the following grants to Eva Sevick: NIH R01 CA128919 and NIH R01 HL092923.

All animal studies were performed in accordance with the standards of the University of Texas Health Science Center (Houston, TX), Department of Comparative Medicine, and Center for Molecular Imaging after review and approval of the protocol by their respective Institutional Animal Care and Use Committee (IACUC) or Animal Welfare Committee (AWC).
1. Preparation of Animals 24 Hr Prior to Imaging
The steps below must be done (as needed) the day before lymphatic im...

<ol>
	<li>Alitalo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphatic+vasculature+in+disease.">The lymphatic vasculature in disease.</a> Nat. Med. 17, 1371-1380 (2011).</li><li>Rasmussen, J. C., Tan, I. C., Marshall, M. V., Fife, C. E., Sevick-Muraca, 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=Lymphatic+imaging+in+humans+with+near-infrared+fluorescence.">Lymphatic imaging in humans with near-infrared fluorescence.</a> Curr. Opin. Biotechnol. 20, 74-82 (2009).</li><li>Rasmussen, J. 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=Human+Lymphatic+Architecture+and+Dynamic+Transport+Imaged+Using+Near-infrared+Fluorescence.">Human Lymphatic Architecture and Dynamic Transport Imaged Using Near-infrared Fluorescence.</a> Transl. Oncol. 3, 362-372 (2010).</li><li>Sevick-Muraca, 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=Translation+of+near-infrared+fluorescence+imaging+technologies:+emerging+clinical+applications.">Translation of near-infrared fluorescence imaging technologies: emerging clinical applications.</a> Annu. Rev. Med. 63, 217-231 (2012).</li><li>Kwon, S., Sevick-Muraca, 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=Noninvasive+quantitative+imaging+of+lymph+function+in+mice.">Noninvasive quantitative imaging of lymph function in mice.</a> Lymphat. Res. Biol. 5, 219-231 (2007).</li><li>Kwon, S., Sevick-Muraca, 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=Mouse+phenotyping+with+near-infrared+fluorescence+lymphatic+imaging.">Mouse phenotyping with near-infrared fluorescence lymphatic imaging.</a> Biomed Opt Express. 2, 1403-1411 (2011).</li><li>Marshall, M. V., 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=Near-infrared+fluorescence+imaging+in+humans+with+indocyanine+green:+a+review+and+update.">Near-infrared fluorescence imaging in humans with indocyanine green: a review and update.</a> The Open Surgical Oncology Journal. 2, 12-25 (2010).</li><li>Davies-Venn, C. A., 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=Albumin-Binding+Domain+Conjugate+for+Near-Infrared+Fluorescence+Lymphatic+Imaging.">Albumin-Binding Domain Conjugate for Near-Infrared Fluorescence Lymphatic Imaging.</a> Mol. Imaging Biol. , (2011).</li><li>Sharma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+lymph+function.">Quantitative imaging of lymph function.</a> Am. J. Physiol. Heart Circ. Physiol. 292, 3109-3118 (2007).</li><li>Kwon, S., Sevick-Muraca, 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=Functional+lymphatic+imaging+in+tumor-bearing+mice.">Functional lymphatic imaging in tumor-bearing mice.</a> J. Immunol. Methods. 360, 167-172 (2010).</li><li>Karlsen, T. V., McCormack, E., Mujic, M., Tenstad, O., Wiig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+quantification+of+lymph+flow+in+mice+and+rats+by+imaging+depot+clearance+of+near-infrared+albumin.">Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin.</a> Am. J. Physiol. Heart Circ. Physiol. 302, 391-401 (2012).</li><li>Zhou, Q., Wood, R., Schwarz, E. M., Wang, Y. J., Xing, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+lymphatic+imaging+demonstrates+the+dynamics+of+lymph+flow+and+lymphangiogenesis+during+the+acute+versus+chronic+phases+of+arthritis+in+mice.">Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice.</a> Arthritis Rheum. 62, 1881-1889 (2010).</li><li>Adams, K. E., 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=Direct+evidence+of+lymphatic+function+improvement+after+advanced+pneumatic+compression+device+treatment+of+lymphedema.">Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema.</a> Biomed. Opt. Express. 1, 114-125 (2010).</li><li>Tan, I. 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=Assessment+of+lymphatic+contractile+function+after+manual+lymphatic+drainage+using+near-infrared+fluorescence+imaging.">Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging.</a> Arch. Phys. Med. Rehabil. 92, 756-764 (2011).</li><li>Lapinski, P. E., 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=RASA1+maintains+the+lymphatic+vasculature+in+a+quiescent+functional+state+in+mice.">RASA1 maintains the lymphatic vasculature in a quiescent functional state in mice.</a> J. Clin. Invest. 122, 733-747 (2012).</li><li>Maus, E. A., 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=Near-infrared+fluorescence+imaging+of+lymphatics+in+head+and+neck+lymphedema.">Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema.</a> Head Neck. 34, 448-453 (2012).</li><li>Galanzha, E. I., Tuchin, V. V., Zharov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+small+animal+mesentery+models+for+in+vivo+flow+cytometry,+dynamic+microscopy,+and+drug+screening.">Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.</a> World J. Gastroenterol. 13, 192-218 (2007).</li><li>Schramm, R., 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=The+cervical+lymph+node+preparation:+a+novel+approach+to+study+lymphocyte+homing+by+intravital+microscopy.">The cervical lymph node preparation: a novel approach to study lymphocyte homing by intravital microscopy.</a> Inflammation research : official journal of the European Histamine Research Society. 55, 160-167 (2006).</li><li>Hall, M. A., 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=Imaging+prostate+cancer+lymph+node+metastases+with+a+multimodality+contrast+agent.">Imaging prostate cancer lymph node metastases with a multimodality contrast agent.</a> Prostate. 72, 129-146 (2012).</li><li>Zhu, B., Sevick-Muraca, 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=Minimizing+excitation+leakage+and+maximizing+measurement+sensitivity+for+molecular+imaging+with+near-infrared+fluorescence.">Minimizing excitation leakage and maximizing measurement sensitivity for molecular imaging with near-infrared fluorescence.</a> J. Innovat. Opt. Health Sci. 4, 301-307 (2011).</li></ol>

Authors have nothing to disclose, but some authors are listed on a patent.

We use a custom, small animal NIRF imaging system to capture images of labeled lymph vessels in mice. To construct movies of lymph movement, 300 or more images are collected. For functional analysis of lymphatics from movies, two or more ROIs are manually drawn along a lymph vessel. The dimensions of the ROIs are kept constant for each vessel and are approximately the diameter of the vessel. While whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, a macrolens for fine...

<table border="1">
 <tbody>
 <tr>
 <td>Solutions, Reagents, and Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Indocyanine green (ICG)</td>
 <td>Patheon Italia S.P.A.</td>
 <td>NDC 25431-424-02</td>
 <td>Reconstitute to 645 &mu;M (5 &mu;g/10 &mu;L)</td>
 </tr>
 <tr>
 <td>Cyclic Albumin Binding Domain(cABD) </td>
 <td>Bachem</td>
 <td>Custom</td>
 <td>Reconstitute to 200 &mu;M (6.8 &mu;g/10 &mu;L) </td>
 </tr>
 <tr>
 <td>IRDye800</td>
 <td>Li-COR</td>
 <td>IRDye 800CW</td>
 <td>Reconstitute according to manufacture's instructions; conjugate with cABD at equilmolar concentrations </td>
 </tr>
 <tr>
 <td>Sterile Water</td>
 <td>Hospira, Inc., Lake Forest, IL </td>
 <td>NDC 0409-4887-10</td>
 <td></td>
 </tr>
 <tr>
 <td>NAIR</td>
 <td>Church &amp; Dwight Co., Inc.</td>
 <td>Local Stores</td>
 <td><a href="http://www.nairlikeneverbefore.com" target="_blank">www.nairlikeneverbefore.com</a> </td>
 </tr>
 <tr>
 <td>Imaging System (components below)</td>
 <td>Center for Molecular Imaging</td>
 <td>N/A</td>
 <td>Custom-built in our laboratories.</td>
 </tr>
 <tr>
 <td>Electron-multiplying charge-coupled device (EMCCD) camera</td>
 <td>Princeton Instruments, Trenton, NJ</td>
 <td>Photon Max 512</td>
 <td></td>
 </tr>
 <tr>
 <td>Nikon camera lens</td>
 <td>Nikon Inc., Melville, NY</td>
 <td>Model No. 1992, Nikkor 28mm</td>
 <td></td>
 </tr>
 <tr>
 <td>Optical filter </td>
 <td>Andover Corp., Salem,NH</td>
 <td>ANDV11333</td>
 <td>Two 830.0/10.0 nm bandpass filters are used in front of lens</td>
 </tr>
 <tr>
 <td>785-nm laser diode</td>
 <td>Intense Ltd, North Brunswick, NJ</td>
 <td>1005-9MM-78503</td>
 <td>500 mW of optical output</td>
 </tr>
 <tr>
 <td>Collimating optics</td>
 <td>Thorlabs, Newton, NJ</td>
 <td>C240TME-B</td>
 <td>Collimates laser output prior to cleanup filter</td>
 </tr>
 <tr>
 <td>Clean-up filter</td>
 <td>Semrock, Inc., Rochester, NY</td>
 <td>LD01-785/10-25</td>
 <td>Removes laser emission in fluorescence band</td>
 </tr>
 <tr>
 <td>Optical diffuser</td>
 <td>Thorlabs, Newton, NJ</td>
 <td>ED1-C20</td>
 <td>Diffuses the laser over the animal</td>
 </tr>
 <tr>
 <td>V++</td>
 <td>Digital Optics, Browns Bay, Auckland, New Zealand</td>
 <td>Version 5.0</td>
 <td>Software used to control camera system and save images to computer. <a href="http://digitaloptics.net/" target="_blank">http://digitaloptics.net/</a> </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Analytic Software Either of the following software packages can be used for image analysis</td>
 </tr>
 <tr>
 <td> ImageJ</td>
 <td>National Institutes of Health, Bethesda, MD</td>
 <td>Most current version available </td>
 <td>Freeware available at <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a></td>
 </tr>
 <tr>
 <td> MATLAB</td>
 <td>MathWorks, Natick, MA</td>
 <td>Version 2008a or later</td>
 <td><a href="http://www.mathworks.com/" target="_blank">http://www.mathworks.com/</a></td>
 </tr>
 </tbody>
</table>

non-invasive optical imaging of the lymphatic vasculature of a mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Example of NIRF Lymphatic Imaging in Mice
When ICG or cABD-IRDye800 is injected ID at the base of the tail of a normal mouse, the lymphatic vasculature between the injection site at the base of the tail and the inguinal lymph node (LN) should be immediately visualized. Shortly after injection (a few seconds to minutes), the lymphatic vessel between the inguinal LN and the axillary LN should be visualized as seen in Figure 2. Since the lymphatics in mice vary from animal to animal...

Watch this Scientific Journal Video about Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse at JoVE.com

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

non-invasive-optical-imaging-of-the-lymphatic-vasculature-of-a-mouse

Research

JoVE Journal

Immunology and Infection

15.9K ビュー.  University of Texas Health Science Center-Houston. 近赤外蛍光（NIRF）を使用して、最近開発されたイメージング技術は、リンパ系の癌転移、免疫応答、創傷修復、および他のリンパ関連疾患に果たす役割の解明に役立つことがあります。