English: Diapedesis of activated 2D2 CD4+ T cells across inflamed cervical spinal cord post-capillary venules during experimental autoimmune encephalomyelitis (EAE). Active EAE was induced as described in Section “Methods.” Laminectomy was performed (described in step 15 of the procedures) on day 22 post-immunization when the mouse showed onset of disease. In vitro activated CD4+ T cells from “2D2-GFP”-mice and 2D2 CD4+ T cells labeled with fluorescent CellTracker CMAC were systemically injected via a carotid catheter before 2P-IVM imaging. A x–y–t time-lapse sequence of a 400 μm × 400 μm scan field at a depth of 47–91 μm and 12 z-stacks with 4 μm spacing is shown. Blood vessels were labeled by injection of Alexa Fluor 594 conjugated anti-endoglin antibody. CD4+ T cells undergoing diapedesis and crawling outside of the vasculature can be observed during 20 min of recording. Due to the unavoidable limited movement of the mouse, the image sequences were cropped at 05:40, 07:00, and 11:20 min. GFP (green, CD4+ T cells), CMAC (blue, fluorescently labeled CD4+ T cell), and Alexa Fluor 594 (red, blood vessels) were excited at 780 nm. Time is shown in minutes and seconds. Scale bar: 50 μm. This video is the source video of which images shown in Figure 7 were taken.