Figure 5

The role of inflammatory induced reticular fibers in naïve T cell migration in healthy and inflamed CNS tissue. (A) Intravital TPLSM reveals inflammation induced reticular fibers which are visualised by second harmonic generation (SHG) at 1110 nm wavelength in the brain stem of EAE affected mice. Naïve T cells partially migrate along SHG structures as demonstrated by cell tracks represented by white arrows exemplarily. For further details see also Additional File 3. (B) To quantify SHG signal and naïve T cell interactions we analyse the co-localisation area of SHG signal (blue) and tdRFP (red). The calculated colocalisation channel (white) was analyzed by determining the duration and the velocity of the tracked contacts (scale bar 10 μm). (C) Interaction patterns were differentiated into short (less than 5 min duration) and long-lasting contacts (equal or longer than 5 min duration,), Most of the contacts were short (79% ± 6; N = 164; white bar) but also long-lasting interactions could be detected (20 ± 6%; N = 38; gray bar) and that these long-lasting contacts are due to migration associated co-localisation as their velocity is > 0.04 μm/s. Data from two independent experiments are shown as percentage of all contacts (± SD). (D) Visualization of the vasculature by IV injection of Rhodamine-dextrane prior to imaging enabled the visualisation of both, the reticular structures (green) and vessels (red), simultaneously revealing that besides the vessel associated SHG signal a large amount of fibers originates directly from the CNS parenchyma outside the perivascular space. 1 unit = 15 μm (E) Intravital TPLSM on brain stem of EAE affected C57BL/6 Thy1.1 CerTN L15 mice which express a FRET based calcium sensor. The Citrulin/Cerulean FRET pair is expressed in neurons and is detected by excitation at 850 nm (left, red) whereas SHG signal was generated by a separated excitation at 1110 nm (middle, green), which does not allow to detect either Citrulin or Cerulean. Images are presented in false colors in order to increase the contrast between both separately detected structures. Merge of both images derived from different excitation modes in the same layer demonstrates that the SHG signal (green) is not co-localised to neuronal structures (red). These images are derived form the same layer at a depth of 60 μm below the surface. Scale bar 30 μm.