Fig. 5.

Two-color, 2P ISIM imaging in a live, anesthetized nematode larva. (a) Ten 2P ISIM volumes were acquired and stitched together to generate a two-color (green, GFP; red, blue-shifted autofluorescence) XY maximum intensity projection of an L2 nematode larva expressing transcriptional reporter psax-3::GFP, which is widely expressed throughout the nervous system. The head of the animal lies to the right, while the tail is located to the left. The yellow rectangle denotes the nerve ring and anterior portion of the nematode gut. Scale bar: 60 μm. (b) Higher-magnification view of the yellow rectangular region in (a), emphasizing nerve cords and nerve ring. Numerous head neurons and ventral cord motor neurons are visible in this view, as well as autofluorescent structures like the terminal bulb of the pharynx and the intestine. Scale bar: 20 μm. (c), (d) Higher-magnification views of yellow rectangular regions in (b). The yellow arrows show both the left and right fascicles of the ventral nerve cord, while the magenta arrow denotes the dorsal nerve cord. A neuronal process connecting the dorsal and ventral nerve cords is visible just anterior to the magenta arrow. In (d), neurons and neuronal processes in the nematode head can be resolved. Scale bar: 4 μm in (c), (d). The green colormap has been saturated in order to highlight dim neurites and subneuronal structures. (e), (f) Axial cuts through the imaging volume, corresponding to magenta and blue dashed lines in (b). Head neurons are visible in both views, while a neurite crossing the dorsal region of the head is denoted by a yellow arrow in (e). Scale bar: 5 μm. Neurites and fasciculating neurites denoted by yellow arrows in (c) and (e) have apparent lateral width <200 nm. All images were deconvolved. See also Media 3.