Name: Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse (Video) | JoVE Journal
Uploaded: 2013-03-08T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn non-invasive near-infrared fluorescence imaging of lymphatic vessels in mice using intradermal dye injection. This method enables visualization of lymphatic architecture and function for studying cancer metastasis and immune response.

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5 minutes

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 imaging takes place.
<ol>
	<li>Place animal in an induction box and sedate with isoflurane.</li>
	<li>Once the animal is in a state of profound anesthesia (monitored with toe-pinch maneuver), place sedated animal on a diaper/fluff pad and position nose in a nose cone connected to isoflurane gas.</li>
	<li>Clip all hair/fur (if any) around the area to be imaged.</li>
	<li>Apply depilatory agent (NAIR) to the clipped area and leave it on the skin for up to 3 min.</li>
	<li>Gently wipe off all depilatory agent with warm, damp gauze or paper towel.</li>
	<li>Gently rinse the skin with warm water and gently dry the area with gauze or paper towel.</li>
	<li>Allow animals to recover on a heating pad or under a heat lamp, and return to their cage.</li>
</ol>
2. Day of Imaging
<ol>
	<li>Reconstitute imaging agent with sterile water, then dilute using sterile, normal (0.85%) saline to achieve 645 &mu;M (5 &mu;g/10 &mu;l) for ICG or 200 &mu;M (6.8 &mu;g/10 &mu;l) for cABD-IRDye800. Keep solutions in dark conditions and use within 6 hr of reconstitution.</li>
	<li>Place animal in an induction box and sedate with isoflurane.</li>
	<li>Once the animal is in a state of profound anesthesia (monitored with toe-pinch maneuver), place sedated animal on its side on a diaper/fluff pad and position nose in a nose cone connected to gas isoflurane.</li>
	<li>Turn off the lights (so the room is dark). If needed, a small desk halogen light can be used for a small amount of light to see injections.</li>
	<li>Using an insulin syringe with a 31-gauge needle, inject ID 5 &mu;l to 50 &mu;l of ICG or cABD-IRDye800 in the dorsal aspect of each hind paw and/or on the left and right side of the base of the tail, depending on the area of interest (see Discussion). Each injected dose may range from 0.083 to 1.25 mg/kg (ICG) or 0.113 to 1.7 mg/kg (cABD-IRDye800). Injection volumes will vary with animal strain and injection site. For athymic mice, the volume of injection can be 5 &mu;l (hind paw) or 10 &mu;l (base of tail). If animal is not under the imaging system for the injection(s), place the animal under the imaging system immediately after the injection(s).</li>
	<li>If no dye uptake is seen in the lymphatics, step 2.5 will need to be repeated as needed per animal protocol.</li>
	<li>Once lymphatics are seen, cover the injection site with black electrical tape or black paper.</li>
	<li>Acquire lymphatic images for up to 1 hr using V++ software and a small animal, NIRF imaging system. (Animals are sedated with isoflurane and respirations are monitored while images are acquiring.) While small animal, NIRF imagers are commercially available, we utilize a customized, small animal NIRF imaging system consisting of a 785-nm laser diode (1005-9mm-78503, Intense, North Brunswick, NJ) outfitted with an aspheric lens (C24TME-B, Thorlabs, Newton, NJ), diffuser (ED1-C20, Thorlabs), and filter (LD01-785/10-25, Semrock, Rochester, NY) to create a uniform excitation field that illuminates the animal at an incident fluence rate of less than 1.4 mW per square centimeter10. An electron multiplying charged-coupled device (EMCCD, PhotonMax512, Princeton Instruments, Trenton, NJ) camera system with two 830-nm filters (AND11333, Andover Corp., Salem, NH) and a 28-mm Nikkor lens (1992, Nikon, Melville, NY) is used to capture lymphatic images with integration times of 200 msec for dynamic imaging and 800 msec for static imaging5. See Figure 1 for system configuration, the Table for additional details of each component, and the Discussion for a brief discussion of key imager properties.</li>
	<li>Allow animals to recover on a heating pad or under a heat lamp and return to their cage, or euthanize.</li>
	<li>Analyze images using ImageJ or MATLAB software. See Figure 6.</li>
</ol>

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<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/pubmed?term=((Lymphatic+imaging+in+humans+with+near-infrared+fluorescence%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Human+Lymphatic+Architecture+and+Dynamic+Transport+Imaged+Using+Near-infrared+Fluorescence%5BTitle%5D)+AND+%22Transl.+Oncol%22%5BJournal%5D)">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/pubmed?term=((Translation+of+near-infrared+fluorescence+imaging+technologies%3A+emerging+clinical+applications%5BTitle%5D)+AND+%22Annu.+Rev.+Med%22%5BJournal%5D)">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/pubmed?term=((Noninvasive+quantitative+imaging+of+lymph+function+in+mice%5BTitle%5D)+AND+%22Lymphat.+Res.+Biol%22%5BJournal%5D)">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/pubmed?term=((Mouse+phenotyping+with+near-infrared+fluorescence+lymphatic+imaging%5BTitle%5D)+AND+%22Biomed+Opt+Express%22%5BJournal%5D)">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/pubmed?term=((Near-infrared+fluorescence+imaging+in+humans+with+indocyanine+green%3A+a+review+and+update%5BTitle%5D)+AND+%22The+Open+Surgical+Oncology+Journal%22%5BJournal%5D)">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/pubmed?term=((Albumin-Binding+Domain+Conjugate+for+Near-Infrared+Fluorescence+Lymphatic+Imaging%5BTitle%5D)+AND+%22Mol.+Imaging+Biol%22%5BJournal%5D)">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/pubmed?term=((Quantitative+imaging+of+lymph+function%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Functional+lymphatic+imaging+in+tumor-bearing+mice%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">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/pubmed?term=((Minimally+invasive+quantification+of+lymph+flow+in+mice+and+rats+by+imaging+depot+clearance+of+near-infrared+albumin%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Heart+Circ.+Physiol%22%5BJournal%5D)">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/pubmed?term=((Near-infrared+lymphatic+imaging+demonstrates+the+dynamics+of+lymph+flow+and+lymphangiogenesis+during+the+acute+versus+chronic+phases+of+arthritis+in+mice%5BTitle%5D)+AND+%22Arthritis+Rheum%22%5BJournal%5D)">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/pubmed?term=((Direct+evidence+of+lymphatic+function+improvement+after+advanced+pneumatic+compression+device+treatment+of+lymphedema%5BTitle%5D)+AND+%22Biomed.+Opt.+Express%22%5BJournal%5D)">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/pubmed?term=((Assessment+of+lymphatic+contractile+function+after+manual+lymphatic+drainage+using+near-infrared+fluorescence+imaging%5BTitle%5D)+AND+%22Arch.+Phys.+Med.+Rehabil%22%5BJournal%5D)">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/pubmed?term=((RASA1+maintains+the+lymphatic+vasculature+in+a+quiescent+functional+state+in+mice%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">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/pubmed?term=((Near-infrared+fluorescence+imaging+of+lymphatics+in+head+and+neck+lymphedema%5BTitle%5D)+AND+%22Head+Neck%22%5BJournal%5D)">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/pubmed?term=((Advances+in+small+animal+mesentery+models+for+in+vivo+flow+cytometry%2C+dynamic+microscopy%2C+and+drug+screening%5BTitle%5D)+AND+%22World+J.+Gastroenterol%22%5BJournal%5D)">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/pubmed?term=((The+cervical+lymph+node+preparation%3A+a+novel+approach+to+study+lymphocyte+homing+by+intravital+microscopy%5BTitle%5D)+AND+%22Inflammation+research+%3A+official+journal+of+the+European+Histamine+Research+Society%22%5BJournal%5D)">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/pubmed?term=((Imaging+prostate+cancer+lymph+node+metastases+with+a+multimodality+contrast+agent%5BTitle%5D)+AND+%22Prostate%22%5BJournal%5D)">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/pubmed?term=((Minimizing+excitation+leakage+and+maximizing+measurement+sensitivity+for+molecular+imaging+with+near-infrared+fluorescence%5BTitle%5D)+AND+%22J.+Innovat.+Opt.+Health+Sci%22%5BJournal%5D)">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><tbody><tr><td>Indocyanine green (ICG)</td><td>Patheon Italia S.P.A.</td><td>NDC 25431-424-02</td><td>Reconstitute to 645 &amp;mu;M (5 &amp;mu;g/10 &amp;mu;L)</td></tr><tr><td>Cyclic Albumin Binding Domain(cABD) </td><td>Bachem</td><td>Custom</td><td>Reconstitute to 200 &amp;mu;M (6.8 &amp;mu;g/10 &amp;mu;L) </td></tr><tr><td>IRDye800</td><td>Li-COR</td><td>IRDye 800CW</td><td>Reconstitute according to manufacture&#039;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 Dwight Co., Inc.</td><td>Local Stores</td><td>&lt;a href=&quot;http://www.nairlikeneverbefore.com&quot; target=&quot;_blank&quot;&gt;www.nairlikeneverbefore.com&lt;/a&gt; </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. &lt;a href=&quot;http://digitaloptics.net/&quot; target=&quot;_blank&quot;&gt;http://digitaloptics.net/&lt;/a&gt; </td></tr><tr><td colspan="4">&lt;em&gt;Analytic Software&lt;/em&gt; 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 &lt;a href=&quot;http://rsbweb.nih.gov/ij/&quot; target=&quot;_blank&quot;&gt;http://rsbweb.nih.gov/ij/&lt;/a&gt;</td></tr><tr><td> MATLAB</td><td>MathWorks, Natick, MA</td><td>Version 2008a or later</td><td>&lt;a href=&quot;http://www.mathworks.com/&quot; target=&quot;_blank&quot;&gt;http://www.mathworks.com/&lt;/a&gt;</td></tr></tbody></table>

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

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...

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 ...

The overall goal of this procedure is to demonstrate how to non-invasively image lymphatics in small animals using a near infrared fluorescence imaging system. This is accomplished by first reconstituting the fluorescent dye to the appropriate concentration immediately before imaging. Next, the sedated animal is injected intradermally with the dye in the location.That will enable visualization of the lymphatic vessels of interest in the final step. Images of the vessels are acquired and then analyzed. Ultimately, near infrared.Fluorescent lymphatic imaging can be used to demonstrate and reveal the function and architecture of the lymphatic vasculature. Though this method can provide insight into the role the lymphatic system plays in healthy animals. It can also be applied in a variety of disease conditions such as cancer metastases, acute and chronic immune response wound healing, and transgenic.Models of lymphatic associated disorders On the day of imaging begin by reconstituting incy and green with sterile water, then dilute with normal saline to a concentration of 645 micromolar. Then after adjusting the nose cone on the sedated animal, turn out the lights if necessary. A small desk Hagen light can be used to see for the injections.The single most difficult aspect of this procedure is injecting the mice into dermally instead of subcutaneously in the correct location for uptake by the lymphatic vessels that are to be visualized. To ensure success, acquire two to three baseline images to confirm the location of the lymphatic vessels in each animal before beginning the experiment. Now use an insulin syringe with a 31 gauge needle to inject 10 microliters of the dye into the left side of the base of the tail to visualize the lymphatics draining from the inguinal region to the axillary region in general.To visualize the left side of the animal, inject in location 5, 6, 9, or 10. To visualize the right side, inject the dye into location 7, 8, 11, or 12 locations, one through four, maybe two inferior on the tail for optimum uptake to visualize lymphatic drainage from the inguinal region to the axillary region. If the animal is not under the imaging system during the injection, immediately place it thereafter.Next, cover the injection site with black paper to help observe the lymphatic vessels using v plus plus software and a small animal near infrared fluorescence imaging system. Acquire lymphatic images for up to one hour. Finally allow the animals to recover on a snuggle heat pad and then return them to their cage while small animal near infrared fluorescent imagers are commercially available.In this video, a customized small animal Nerf imaging system consisting of a 785 nanometer laser diode outfitted with an aspheric lens diffuser and filter to create a uniform excitation field that illuminates the animal at an incident fluence rate of less than 1.4 milliwatts per square centimeter was used and electron multiplying charge couple device camera system with 2 830 nanometer filters and a 28 millimeter nyco lens is used to capture lymphatic images with integration times of 200 milliseconds for dynamic imaging and up to 800 milliseconds for static imaging of fluorescent lymph. When incy in green or cyclic albumin binding domain peptide IR dye 800 is injected intradermally 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 should be immediately visualized within a few seconds to minutes after the injection.The lymphatic vessel between the inguinal lymph node and the axillary lymph node should be visualized as seen here since the lymphatics in mice vary from animal to animal as they do in humans. Variations in architecture between animals may be seen as demonstrated in this figure. These panels show images obtained on the day of injection one, and these panels show images obtained two days later on the day of injection two, using the same mice and imaging protocol.While the lymphatic architecture varies between mouse 1 24 and 1 28, the images obtained using the near infrared fluorescence imager are consistent for each mouse. On days one and three, when the imaging agent is injected intradermally on the dorsal aspect of the hind paw of a normal mouse, two lymphatic vessels can be visualized draining to the popliteal lymph node as shown here. In some cases, it is difficult to distinguish both vessels because of their proximity as illustrated in this magnification of the image of the animal's hind limb.For the representative mouse shown here, 10 microliters of ICG was injected in the dorsal aspect of the left hind paw as well as into the left side of the tail base. At times, visualization of the lymphatics is delayed most commonly due to the injection being administered subcutaneously instead of intradermally. When subcutaneous injections are given, lymphatic transport may not be immediately visualized as illustrated in this image because of the additional time required for the dye to reach and be taken up by the lymphatic capillaries in the skin.Thus, it is important to take care the dye be injected intradermally not subcutaneously. On occasion, abnormal lymphatic vessels are observed. For example, in this image on the non-injured side of the animal, the inguinal lymph node can be visualized as well as the relatively straight EENT lymph vessel draining up toward the axillary lymph nodes On the mouse's injured side.However, the normal lymph vasculature was interrupted due to wounding and appears abberant due to tissue repair acquired images of the visualized lymphatic vessels can be loaded into Image J or MATLAB for data analysis. As shown in these images, constant area circular regions of interest are selected or drawn along the entire length of the fluorescent lymphatic vessel. In human and animal lymphatic imaging, a region of interest is selected such that its diameter is approximately the diameter of the image of the fluorescent vessel.The mean fluorescence intensity within each region of interest is then plotted as a function of imaging time to assess the propulsive velocity and frequency of the packets of dia laden lymph propelled along the lymphatic vessels. The perturbations in fluorescence intensity across the ROIs represent a lymphatic pulse propagating through the regions of interest and are parallel to the arrows to assess the lymphatic propagation velocity and frequency of lymphatic propulsion. Two regions of interest with clearly defined maxima or fluorescent intensity variations representing the propagation of packets of lymph are selected and their fluorescent intensity profiles are plotted as can be observed in these two graphs.The propagation velocity is computed by taking the ratio of the distance between the two regions of interest and the transit time for a packet of lymph to propagate between them. By assessing the number of fluorescent pulses or packets arriving at a single region of interest per time, the contractile frequency is computed After its development. This technique paved the way for researchers to non-invasively and longitudinally elucidate the role of the lymphatic system in a variety of diseases in health conditions.

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

Research

JoVE Journal

Immunology and Infection

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1.  Here, we demonstrate adoptive transfer of  T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Intravital Microscopy of the Inguinal Lymph Node

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions.  Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Intravital Imaging of the Mouse Popliteal Lymph Node

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from naïve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy

A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented.

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

The manuscript demonstrates an innovative method for in vivo study of the contractility of cervical lymphatic vessels and lymph flow in mice for testing various physiological functions of cervical lymphatic vessels (cLVs) and the therapeutic strategies for modulation of the lymphatic regulatory mechanisms of brain drainage and clearance.

Reproducible Setup for In vivo Two-photon Imaging of the Contractility of Cervical Lymphatic Vessels and Lymph Flow in Mice

This protocol assesses lymphatic drainage after fracture by combining indocyanine green(ICG) near-infrared imaging with ultrasound to quantify plantar ICG clearance and popliteal lymph node enlargement in mice.

The Assessment of Lymphatic Drainage Using ICG Near-Infrared Imaging and Ultrasound After Fracture

Learn non-invasive near-infrared fluorescence imaging of lymphatic vessels in mice using intradermal dye injection. This method enables visualization of lymphatic architecture and function for studying cancer metastasis and immune response.

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

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