Scanning Electron Microscopy of Strongyloxea

Strongyloxea from T. aurantia sponges were received dried and separated from the surrounding spongin. The Sxa were first examined using a polarized light microscope (Nikon Ci Pol). Intact, undamaged Sxa were mounted to aluminum stubs using conductive carbon tape. The mounted Sxa were sputter coated with approximately 10 nm of carbon and then imaged with a scanning electron microscope (FEI Helios, or LEO 1530 VP) at roughly 500X magnification. At this magnification, the field of view was roughly 250 μm × 200 μm in the FEI Helios (130 μm × 90 μm in the LEO 1530 VP). Therefore, a complete image of a Sxa consisted of 7–14 overlapping frames. These frames were aligned and stitched together to make a single composite image using a Fourier transform-based phase correlation method implemented in ImageJ58 (link). A representative composite image is shown in Supplementary Fig. S1.