Figure 4: Filament tracing in optically cleared peripheral nerve and skeletal muscle. (A) Fluorescently labeled neurovascular system in a rat common peroneal nerve segment. Segmentations as seen in B and C can compartmentalize the blood vessel network in inter-branch segments and thereby allow for detailed analysis of the blood vessel diameter (D), the distance between two branching sites (E) or the angle of the vascular axis (F). (G) Similar fluorescence-based filament tracing techniques can be used to map the intermuscular nerve branches in the peroneus longus muscle of a transgenic Thy-1 GFP+ rat with green-fluorescent axons (scale bar). (H) Intramuscular nerve branch in higher magnification (scale bar) (I) Neuromuscular junctions stained with fluorescently conjugated α-bungarotoxin (red) can be mapped via image segmentation (white overlay in J) in cleared muscle and quantitatively analyzed for their three-dimensional surface area (K) to determine structural changes related to development or disease. Of note, GFP-positive axon terminal innervating the NMJs are not visible with the applied imaging parameters. Scale bars: 100 μm in A–C, I, and J, 1000 μm in C, 50 μm in H. NMJ: Neuromuscular junction; Q: quartile; SD; standard deviation.