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 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.
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.
The authors have nothing to disclose.
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.
Reagent | Company | Catalogue Number |
Isoflurane, USP | Baxter Healthcare Corporation | NDC 10019-773-60 |
Vetbond | 3M | 1469SB |
Nair – hair removal lotion | Nair | |
PBS, 1X | Cellgro | 21-040-CV |
Heating pad | Watlow | |
Heating Pad Controller | Watlow | |
Air and O2 | Airgas | |
Temperature probe | Harvard Apparatus | |
Tweezers Dumont #5 | World Precision Instruments, Inc | 14101 |
Forceps, Graefe Iris, 7 cm curved | World Precision Instruments, Inc | 14141 |
Scissors | Roboz Surgical Instrument Co., Inc | RS-5880 |
Cover of 100×20 mm glass cell culture dish | Corning | 70165-102 |
Cover of 100×20 mm polystyrene cell culture dish | Corning | 430167 |
Betadine Solution (10% Povidine-Iodine Topical Solution) | Purdue Products, L.P. | NDC 67618-150-08 |