Intravital multiphoton imaging of metastases. (A–F) Still images from the time-lapse videos (Videos 1–4) obtained by in vivo multiphoton microscopy of metastases from MDAcl.13 tumors. (A–D) Metastasis at the junction of the afferent lymphatic vessel and the lymph node. (A) Maximum intensity projection image showing an afferent lymphatic vessel leading into the SCS (junction is indicated by *). Collagen fibers of the lymph node capsule and the lymphatic vessel wall are visualized by SHG (blue). SCS contains a large cluster of tumor cells (GFP, green). Evan’s Blue tracer (white) labels the lymphatic vessel and the SCS. Some LN cells have taken up the Evan’s blue dye and appear white. Blood vessels are labeled by i.v. injection of 2 MDa rhodamine dextran (red). (B) Higher magnification of the boxed area in A (25 µm wide) showing a tumor cell with an extended and retracted protrusion at different time points as indicated in minutes. (C and D) Tumor cells (GFP, green) in the SCS (white) in the single plane 35 µm beneath the LN capsule. Leukocytes in the SCS appear black on the white background. (C) The pink line delineates movement of one leukocyte tracked over 61 min (distance traveled: 210 µm). (D) Higher magnification view showing one tumor cell which changed position (arrows) and another tumor cell in the proximity which remained stationary (arrowheads). (E) Tumor cell clusters in the collecting, afferent lymphatic vessel remain in the same position within the 65-min observation period. Lymphatic valve (dashed lines) is shown closed and partially open at indicated time points. Arrow points to a migrating cell. (F) Higher magnification of an area in E. A single cell is shown (arrows, dotted lines) which migrates into a different plane within the 20 min from the onset of cell shape change. See also Videos 1–4. LV, lymphatic vessel. Bars: (A, C, and E) 100 µm; (D and F) 30 µm.