This work is supported by grants from NCI 1R01CA154656 , NIAID 1R21AI092299, Cancer Research Institute, St. Baldrick's Foundation, Dana Foundation, Gabrielle's Angel Foundation, and Hyundai Motors of America "Hope-on-Wheels" Program.

1. Mouse Holder Assembly
<ol>
	<li>Overlay the cover of a 100-mm glass Petri dish on top of a 100-mm plastic Petri dish lid facing down. The glass should be barely touching the center point of the plastic dish. Using the glass as a stencil, trace a mark onto the plastic dish.</li>
	<li>Use a hand drill (i.e. Dremel) to remove a portion of a 100-mm plastic dish lid to create a crescent-shaped platform. This crescent-shaped plastic lid will serve as a support for the upper torso of the mouse (Figure 1a). Drill two holes on the crescent-shaped lid to secure the gas mask with a metal twist tie. Secure the plastic lid to the edge of the glass Petri dish using super glue or an equivalently strong adhesive (Figure 1b).</li>
	<li>Glue the lids of two domed-cap PCR tubes (cut at the hinge) 1 cm apart to the center of the bottom dish for the purpose of anchoring popliteal skin flaps.</li>
</ol>
2. Mouse Preparation
Note: With sufficient practice, one should be able to perform the mouse preparation and surgical steps in 20-30 minutes.
<ol>
	<li>Anesthetize the mouse with isoflurane (2 to 2.5% for induction, 1.5 to 2% for surgery / imaging) admixed in 1:1 O2 : air mixture at a flow rate of 1L/min using an IACUC- approved procedure. Once the mouse is anesthetized, secure a gas mask over the nose with tape. Determine if the mouse is fully anesthetized by the lack of response to toe and/or tail pinches. The level of isoflurane may be adjusted accordingly to ensure the animal is fully sedated, with a steady, non-labored respiratory rate between 60-80 breaths/min.</li>
	<li>Use an electric trimmer to remove hair on the right hind leg and inguinal area of the mouse.</li>
	<li>Brush away loose hair and gently apply a modest coat of Nair lotion onto the shaven area with a cotton swab. After one minute of initial application, remove the Nair and clean the exposed skin with a damp paper towel. Make sure the mouse is clean and dry before proceeding.</li>
	<li>Make a small 2 to 3 mm incision with scissors at the right knee to expose the extensor tendon.</li>
	<li>Apply Vetbond along the center of the mouse holder where the mouse body and leg will be positioned. Carefully secure the mouse onto the holder, with the right knee down, to expose the right popliteal fossa. Secure the right knee tendon with Vetbond in between the 2 skin flap holders to help stabilize the imaging field.</li>
	<li>Stretch and tape the arms and left leg to the upper platform of the holder.</li>
	<li>Use a twist tie to secure the gas mask in place.</li>
	<li>Place the holder under the dissection microscope. The LN must remain absolutely still, independent of the breathing motion of the mouse. Therefore, precautions must be taken while performing the surgery to optimize the stability of the leg. Determine the position of the tail (typically above the head) that will contribute to the greatest stability to the right leg and tape the tail down.</li>
</ol>
3. Surgery
<ol>
	<li>While under the dissection scope, maintain mouse body temperature using a space heater or a heating pad. For successful imaging of the popliteal LN, it is critical to sustain proper body temperature throughout the surgery as well as to preserve tissue moisture by constantly applying warm PBS to the exposed tissue.</li>
	<li>Sterilize the skin with Betadine. Using sterile scissors, make a midline incision through the skin at the right mid-calf, and continue cutting vertically up to the superior portion of the right thigh.</li>
	<li>Make two horizontal skin incisions at the top of the vertical incision line to create skin flaps on either side.</li>
	<li>Retract and glue down both skin flaps with Vetbond. Pulling the skin taut prior to applying Vetbond will further promote leg stability; however, make sure that the skin tension does not occlude blood flow (i.e. changes in the vessel color or diameter). Continue to glue down other areas of the skin to ensure stability of the leg before exposing the LN. The size of the mouse will determine how much extra skin is needed to glue down. Typically, bigger mice will require more skin to be glued to the holder.</li>
	<li>The LN should lie within the popliteal fossa either to the right or left of the popliteal vein, depending on the placement of the mouse on the holder. Carefully separate the LN from surrounding adipose tissues and muscles using micro-dissecting tweezers and forceps. To minimize bleeding and trauma, use the splaying techniques with micro-dissecting tweezers to separate tissues. Carefully expose the popliteal LN without jeopardizing the integrity of the afferent and efferent blood vessels and the afferent lymphatic vessels.</li>
	<li>For added tissue stability, a square cover glass may be positioned over the moist LN, just barely touching the LN while avoiding vessel occlusion. The cover glass may be secured with modeling clay on either side of the mouse.</li>
	<li>Fluorescent vessel dyes of various sizes (e.g. TRITC-dextran, minimum of 70kDa) may be introduced intravenously at this point to help highlight the structural relationship and integrity of the LN during imaging.</li>
</ol>
4. 2-Photon Imaging Acquisition**
<ol>
	<li>Once the LN is adequately exposed and the stability is achieved, promptly transfer the entire mouse holder assembly onto the microscope stage fitted with a programmable temperature feedback controlled heating pad in an environmental microscope chamber kept at 37°C. Add enough sterile warm (37°C) PBS or HBSS to submerge the LN. The volume will change depending on the size and placement of the mouse. Alternatively, warm PBS or HBSS may be applied directly to the dissected LN through a fine glass pipette fitted to a peristaltic pump and taped to the column of the immersion lens objective. The flow of the liquid should be such that a stable water column can be maintained between the lens and the tissue.</li>
	<li>Maintain the PBS or HBSS temperature at 37°C using a heating pad with a feedback probe. A separate temperature probe should be placed in the mouse holder to confirm the temperature of the PBS in the range of 36.5 to 37.5°C. A rectal probe may also be used to monitor core body temperature of the experimental mouse throughout the imaging session.</li>
	<li>Use an epi-fluorescent lamp to help guide LN placement under the objective. Acquire fluorescent image stacks using time intervals that are appropriate for the desired cellular interaction (typically 10 seconds to 1 minute in between each xyz image stack).</li>
	<li>Monitor the status of the animal in the microscope chamber frequently by visual inspection or by using an animal monitoring system. Determine if the mouse is fully anesthetized by the lack of response to toe and/or nail pinches, and a steady, non-labored respiratory rate between 60-80 breaths/min. The level of isoflurane may be adjusted accordingly to ensure that the animal is fully sedated. With proper monitoring and hydration, animals can be imaged for 4-6 hours, or possibly longer.</li>
	<li>After the imaging experiment, euthanize the animal in a CO2 chamber using IACUC-approved euthanasia protocol. The animal should not be allowed to emerge from anesthesia prior to euthanasia.</li>
</ol>
** This surgical procedure may also be useful for other forms of intravital imaging other than 2P-LSM.
5. Representative Results
Various circulating immune cells are recruited to the LN at different rates following adoptive transfer. For CD4+ and CD8+ lymphocytes, these cells begin to arrive in the LN through the high endothelial venule (HEV) minutes after i.v. transfer with substantial numbers arriving in the popliteal LN after 2 to 4 hours6,17-18. For B cells, a substantial number will accumulate after 8 to 24 hours19. Activated DCs should start to appear in the draining popliteal LN 8 to 16 hours following footpad injection3,11,16-17. Figure 2a shows that even without other landmarks, structures such as B cell follicles can be discerned easily by the round spherical cell accumulation visible under 2P-LSM19. Using an endogenous fluorescent reporter such as the ubiquitin-GFP splenocytes (Figure 2, Supplemental Videos 1 and 2), one can track these lymphocyte migrations and behaviors for days up to a week under non-stimulatory physiologic conditions. With multi-channel high sensitivity detectors, it is possible to acquire a multiplex imaging dataset that encompasses structural information as well as interaction dynamics among multiple cellular partners17,19.
When the surgical techniques are properly executed and the environmental conditions carefully monitored, lymphocytes should exhibit characteristic migration speed, as demonstrated in Figure 2c, 2d and elsewhere13-14,16. Lymphocytes may also exhibit differences in migration speed depending on the sub-regions of the LN undergoing imaging, so additional landmarks such as blood vessels (as highlighted by the introduction of vessel dyes) will help to determine the overall imaging quality (i.e. proper temperature control, minimal trauma to LN, etc.)3,6,20.
<img alt="Surgical apparatus setup for animal study; steps, equipment, and anatomical detail display." src="/files/ftp_upload/3720/3720fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1."> 
Figure 1. Construction of a mouse holder for mouse popliteal LN Imaging. a) Schematics of mouse holder assembly; b) Representative intravital mouse preparation; c) Completed mouse holder assembly; d) Close-up views of the popliteal LN after surgical exposure.
<img alt="B cell follicle microscopy and speed distribution graph for immune cell movement analysis." src="/files/ftp_upload/3720/3720fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2."> 
Figure 2. Migration analysis of GFP+ lymphocytes in the popliteal LN. (a) 3D snapshot taken from 2P-LSM imaging sequence of the popliteal LN in a C57BL/6 recipient mouse adoptively transferred with 1x107 GFP+ lymphocytes 1 day prior to imaging. Dash line denotes the border of B cell follicles; (b) Tracks of lymphocyte migration during 1 hour of continuous imaging; c) Distribution of overall lymphocyte migration speed. Mean speed = 10.04 ± 4.26 μm/min (total of 15,125 tracks analyzed); d) Differential cellular migration speed distribution of cells found in the B cell follicle (open bars; mean speed = 8.79 ± 3.90 μm/min; total 1,525 tracks analyzed) and T cell zone (closed bars; mean speed = 13.77 ± 5.93 μm/min; total of 1,250 tracks analyzed). Scale bar = 50 μm.
Supplemental Video 1. Time-lapse intravital 2P-LSM imaging of a mouse popliteal LN as described in Figure 2. A total of 1x107 lymphocytes were isolated from an ubiquitin-GFP+ donor mouse and adoptively transferred intravenously into a C57BL/6 recipient mouse 24 hours before imaging. A series of xy (750 μm x 750 μm) fluorescence images were taken through fixed z stacks (5 μm steps, 13 steps) to yield an xyz imaging stack (750 μm x 750 μm x 65 μm), which was repeated every 20 seconds for a total of 60 minutes, resulting in an xyzt imaging sequence for speed analysis (Figures 2c, 2d). Playback speed = 450x. Scale bar = 50 μm. Time stamp = min:sec. <a href="https://www.jove.com/files/ftp_upload/3720/Huang_Jove_video1.avi" target="_blank"> Click here to watch supplemental video</a>.
Supplemental Video 2. Zoomed-in view of the imaging sequence in Supplemental Video 1 at the B cell follicle - T cell zone border. Playback speed = 450x. Scale bar = 25 μm. Time stamp = min: sec. <a href="https://www.jove.com/files/ftp_upload/3720/Huang_Jove_video2.avi" target="_blank"> Click here to watch supplemental video</a>.

<ol><li>Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-Cell+activation+by+dendritic+cells+in+the+lymph+node%3A+lessons+from+the+movies%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">T-Cell activation by dendritic cells in the lymph node: lessons from the movies.</a> Nat. Rev. Immunol. 8, 675-684 (2008).</li>
<li>Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+friends+in+out-of-the-way+places%3A+how+cells+of+the+immune+system+get+together+and+how+they+conduct+their+business+as+revealed+by+intravital+imaging%5BTitle%5D)+AND+%22Immunol.+Rev%22%5BJournal%5D)">Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging.</a> Immunol. Rev. 221, 163-181 (2008).</li>
<li>Huang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Illuminating+the+landscape+of+in+vivo+immunity%3A+insights+from+dynamic+in+situ+imaging+of+secondary+lymphoid+tissues%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Illuminating the landscape of in vivo immunity: insights from dynamic in situ imaging of secondary lymphoid tissues.</a> Immunity. 21, 331-339 (2004).</li>
<li>Stefanova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-recognition+promotes+the+foreign+antigen+sensitivity+of+naive+T+lymphocytes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Self-recognition promotes the foreign antigen sensitivity of naive T lymphocytes.</a> Nature. 420, 429-434 (2002).</li>
<li>Stefanova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+role+of+self-recognition+in+T+cell+responses+to+foreign+antigen%5BTitle%5D)+AND+%22Immunol.+Rev%22%5BJournal%5D)">On the role of self-recognition in T cell responses to foreign antigen.</a> Immunol. Rev. 191, 97-106 (2003).</li>
<li>Bajenoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibroblastic+reticular+cells+guide+T+lymphocyte+entry+into+and+migration+within+the+splenic+T+cell+zone%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone.</a> J. Immunol. 181, 3947-3954 (2008).</li>
<li>Celli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+the+dynamics+of+T+cell-dendritic+cell+interactions+in+vivo%5BTitle%5D)+AND+%22Immunol.+Rev%22%5BJournal%5D)">Decoding the dynamics of T cell-dendritic cell interactions in vivo.</a> Immunol. Rev. 221, 182-187 (2008).</li>
<li>Mempel, T. R., Junt, T., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rulers+over+randomness%3A+stroma+cells+guide+lymphocyte+migration+in+lymph+nodes%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Rulers over randomness: stroma cells guide lymphocyte migration in lymph nodes.</a> Immunity. 25, 867-869 (2006).</li>
<li>Worbs, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CCR7+ligands+stimulate+the+intranodal+motility+of+T+lymphocytes+in+vivo%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">CCR7 ligands stimulate the intranodal motility of T lymphocytes in vivo.</a> J. Exp. Med. 204 (3), 489-495 (2007).</li>
<li>Bajenoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((byways+and+breadcrumbs%3A+directing+lymphocyte+traffic+in+the+lymph+node%5BTitle%5D)+AND+%22Trends+Immunol%22%5BJournal%5D)">byways and breadcrumbs: directing lymphocyte traffic in the lymph node.</a> Trends Immunol. 28, 346-352 (2007).</li>
<li>Mempel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (2004).</li>
<li>Mempel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues%5BTitle%5D)+AND+%22Curr.+Opin.+Immunol%22%5BJournal%5D)">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr. Opin. Immunol. 16, 406-417 (2004).</li>
<li>Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 2604-2609 (2003).</li>
<li>Sumen, C., Mempel, T. R., Mazo, I. B., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+microscopy%3A+visualizing+immunity+in+context%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Intravital microscopy: visualizing immunity in context.</a> Immunity. 21, 315-329 (2004).</li>
<li>von Andrian, U. H., Mempel, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Homing+and+cellular+traffic+in+lymph+nodes%5BTitle%5D)+AND+%22Nat.+Rev.+Immunol%22%5BJournal%5D)">Homing and cellular traffic in lymph nodes.</a> Nat. Rev. Immunol. 3, 867-878 (2003).</li>
<li>Bousso, P., Robey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+CD8%2B+T+cell+priming+by+dendritic+cells+in+intact+lymph+nodes%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">Dynamics of CD8+ T cell priming by dendritic cells in intact lymph nodes.</a> Nat. Immunol. 4, 579-585 (2003).</li>
<li>Castellino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines+enhance+immunity+by+guiding+naive+CD8%2B+T+cells+to+sites+of+CD4%2B+T+cell-dendritic+cell+interaction%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.</a> Nature. 440, 890-895 (2006).</li>
<li>Halin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+lymphocyte+trafficking%5BTitle%5D)+AND+%22Annu.+Rev.+Cell+Dev.+Biol%22%5BJournal%5D)">In vivo imaging of lymphocyte trafficking.</a> Annu. Rev. Cell Dev. Biol. 21, 581-603 (2005).</li>
<li>Qi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrafollicular+activation+of+lymph+node+B+cells+by+antigen-bearing+dendritic+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells.</a> Science. 312, 1672-1676 (2006).</li>
<li>Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+extended+vision+for+dynamic+high-resolution+intravital+immune+imaging%5BTitle%5D)+AND+%22Semin.+Immunol%22%5BJournal%5D)">An extended vision for dynamic high-resolution intravital immune imaging.</a> Semin. Immunol. 17, 431-441 (2005).</li>
</ol>

The authors do not have anything to disclose.

Recent advances in high-resolution in situ imaging techniques, especially 2P-LSM, have been accompanied by a growing interest in the study of dynamic cellular behavior in vivo. The 4D imaging technique on the popliteal LN of a live mouse allows such analyses in the dynamic behavior of immune cells within the undisrupted tissue micro-environment. The use of 2P-LSM with multiple detectors spanning the entire visible spectrum permits simultaneous imaging data collection of multiple cell populations. This can now be achieved through the use of in vivo cell-specific fluorescent reporter mice (e.g. Ubiquitin-eGFP, -RFP, or -eCFP) combined with the use of adoptive transfer of differentially labeled cell populations using organic fluorescent cell dyes (e.g. CFSE, SNARF-1, and Cell Tracker Orange) to examine cellular mechanisms and function within the LN. In addition to the direct observation of interactions between differentially tracked cell populations, the multiplex imaging dataset can undergo further analysis with commercially available imaging processing software programs (e.g. Imaris, BitPlane Inc.) to further elucidate cell behavior and function. A broad array of possibilities exists to study cellular interaction mechanisms using these in vivo and in silico techniques.
The main limitation of the experimental approach described here is the technical complexity inherent in the surgery approach. This technique requires rigorous training to become familiar with the relevant anatomy and the precise technical procedures and skills as required by this protocol. Further complicating factors include the difficulty in minimizing tissue damage during LN exploration, optimizing tissue stability during imaging, and preventing thermal and laser injury to the LN before and during imaging experimentation. Perturbation to any of these factors will result in less-than-optimal lymphocyte motility and will therefore interfere with proper interpretation of resulting imaging data analyses.

<table><tbody><tr><td>Isoflurane, USP</td><td>Baxter Internationl Inc.</td><td>NDC 10019-773-60</td><td></td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Nair - hair removal lotion</td><td>Nair</td><td></td><td></td></tr><tr><td>PBS, 1X</td><td>Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Heating pad </td><td>Watlow</td><td></td><td></td></tr><tr><td>Heating Pad Controller</td><td>Watlow</td><td></td><td></td></tr><tr><td>Air and O&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td></td></tr><tr><td>Temperature probe</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Tweezers Dumont #5</td><td>World Precision Instruments, Inc.</td><td>14101</td><td></td></tr><tr><td>Forceps, Graefe Iris, 7 cm curved</td><td>World Precision Instruments, Inc.</td><td>14141</td><td></td></tr><tr><td>Scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-5880</td><td></td></tr><tr><td>Cover of 100x20 mm glass cell culture dish</td><td>Corning</td><td>70165-102</td><td></td></tr><tr><td>Cover of 100x20 mm polystyrene cell culture dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>Betadine Solution (10% Povidine-Iodine Topical Solution)</td><td>Purdue Products L.P.</td><td>NDC 67618-150-08</td><td></td></tr></tbody></table>

intravital imaging of the mouse popliteal lymph node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Watch this Scientific Journal Video about Intravital Imaging of the Mouse Popliteal Lymph Node at JoVE.com

Intravital Imaging of the Mouse Popliteal 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.

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

intravital-imaging-of-the-mouse-popliteal-lymph-node

Research

JoVE Journal

Immunology and Infection

28.3K Views.  Case Western Reserve University. Recente ontwikkelingen in de 2-foton microscopie hebben real-time ingeschakeld In Situ beeldvorming van levende weefsels in diermodellen, en zodoende het ons vermogen om cellulaire gedrag te onderzoeken, zowel in fysiologische en pathologische omstandigheden. Hier hebben we een overzicht van de voorbereidingen die vereist zijn om intravitale beeldvorming van de muis popliteale lymfeklieren uit te voeren.

Video: Intravital Imaging of the Mouse Popliteal Lymph Node

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

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.

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

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

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

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.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Biology

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.

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 ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

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

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

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.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

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

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

Medicine

Popliteal Vascular Lymph Node Resection in the Rabbit Hindlimb for Secondary Lymphedema Induction

Lymphedema is a common condition often associated with cancer and its treatment, which leads to damage to the lymphatic system, and current treatments are mostly palliative rather than curative. Its high incidence among oncologic patients indicates the need to study both normal lymphatic function and pathologic dysfunction. To reproduce chronic lymphedema, it is necessary to choose a suitable experimental animal. Attempts to establish animal models are limited by the regenerative capacity of the lymphatic system. Among the potential candidates, the rabbit hindlimb is easy to handle and extrapolate to the human clinical scenario, making it advantageous. In addition, the size of this species allows for better selection of lymphatic vessels for vascularized lymph node resection.
In this study, we present a procedure of vascular lymph node resection in the rabbit hindlimb for inducing secondary lymphedema. Anesthetized animals were subjected to circumferential measurement, patent blue V infiltration, and indocyanine green lymphography (ICG-L) using real-time near-infrared fluorescence, a technique that allows the identification of single popliteal nodes and lymphatic channels. Access to the identified structures is achieved by excising the popliteal node and ligating the medial and lateral afferent lymphatics. Special care must be taken to ensure that any lymphatic vessel that joins the femoral lymphatic system within the thigh without entering the popliteal node can be identified and ligated. 
Postoperative evaluation was performed at 3, 6, and 12 months after induction using circumferential measurements of the hindlimb and ICG-L. As demonstrated during follow-up, the animals developed dermal backflow that was maintained until the 12th month, making this experimental animal useful for novel long-term evaluations in the management of lymphedema. In conclusion, the approach described here is feasible and reproducible. Additionally, during the time window presented, it can be representative of human lymphedema, thus providing a useful research tool.

Here, the surgical induction of stable acquired lymphedema in the rabbit hindlimb is described. This experimental animal can be used to further investigate the effect of lymphedema treatment by microsurgical techniques.

Lymphedema is a common condition often associated with cancer and its treatment, which leads to damage to the lymphatic system, and current treatments are ...

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

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

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

Intravital Imaging of the Mouse Popliteal Lymph Node

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