Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.
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This work was supported by the NIH under award nos. R00CA137167 (T.P.P.), DP2OD008780 (T.P.P.), R01CA163528 (B.J.V.) and R01HL128168 (T.P.P.). Research reported in this publication was supported in part by the Center for Biomedical OCT Research and Translation through grant no. P41EB015903 (B.J.V.), awarded by the National Institute of Biomedical Imaging and Bioengineering of the NIH. National Cancer Institute Federal Share of Proton Income grant CA059267 (T.P.P., B.J.V.) and the Massachusetts General Hospital Executive Committee on Research ISF (T.P.P.) also supported this work. We thank J. Kahn, E. Beech and C. Cui for outstanding technical support.
Integrated supplementary information
The CLNW is used to visualize lymph node neutrophil and monocyte movement (green) in LysM-GFP transgenic mice. Intravenously injected tetramethylrhodamine-dextran dye is used to trace the vasculature (red). Tetramethylrhodamine-dextran extravasation is visible in the top left quadrant. Collagen (blue) is acquired by SHG imaging. The time-lapse MPM movie was taken over 90 min at 1× zoom, stack depth ~45 μm (z-projected using ImageJ software), with a 1.05 NA 25× objective, 512 × 512 pixels, 16-bit. Resolution not enhanced. Scale bar, 50 μm. This experiment was approved by the IACUC at MGH.
In this 30-min MPM movie, lymph node neutrophil and monocyte movement (green) is visualized in LysM-GFP transgenic mice. These cells can be seen flowing, rolling and extravasating in a lymph node high-endothelial venule (red), a specialized blood vessel in the lymph node used for lymphocyte migration. The blood vessel imaged is not directly (~130μ) below the cover slip, causing a reduced signal-to-noise ratio in the video but demonstrating the viability of MPM in deeper-situated regions of interest. Tetramethylrhodamine-dextran dye is used to trace the vasculature (red). Collagen (blue) is acquired by SHG imaging. Images acquired at 2× zoom, stack depth ~20 μm (z-projected using ImageJ software), with a 1.05 NA 25× objective, 256 × 256 pixels, 16-bit. Resolution not enhanced. Scale bar, 25 μm. This experiment was approved by the IACUC at MGH.