Synchrotron imagery of phosphatized eggs in Waptia cf. W. fieldensis from the middle Cambrian (Miaolingian; Wuliuan) Spence Shale of Utah

The Waptia cf. W. fieldensis Walcott, 1912 specimen (KUMIP 314032) (Fig. 1.2–1.3, 2.4–2.7) was collected by the Gunther Family at Miners Hollow (Section 14, T10N, R02W; 41.6023°N, 112.0334°W), Wellsville Mountains, Langston Formation, Spence Shale, Utah, United States (Kimmig et al., 2019, p. 610, fig. 1). The specimen was photographed with a Canon EOS 5D Mark II (f/6.3, 1/100 sec, ISO 2500, 100 mm focal length) using different illumination techniques (Fig. 1). Dimensions of eggs were measured using Dragonfly version 2021.1 for Windows (Object Research Systems Inc., Montreal, Canada, 2021), where max length and diameter measurements were taken from the dorsal point of view to keep the orientation consistent (Table 1). Portions of eggs cropped out of the synchrotron images were estimated based on Figure 2.7 using FIJI ImageJ software for Windows (Schindelin et al., 2012). Synchrotron images were taken at beamline ID 19 of the European Synchrotron Radiation Facility, Grenoble, France, to investigate the ultrastructural details of the eggs. One of the eggs (referred to as Egg One) (Fig. 1.7, 3.2–3.4) became loose and was analyzed separately from the matrix. Egg One was imaged using 26.5 keV monochromatic beam at 0.65 μm resolution. All other eggs were scanned using 110 keV monochromatic beam at 0.64–0.66 μm resolution. Synchrotron images were converted from .TIFF to grayscale .JPEG using the FIJI ImageJ software for Windows (Schindelin et al., 2012) batch convert tool to accommodate hardware limitations. The converted data were volume-rendered in Dragonfly version 2021.1 (Object Research Systems Inc., Montreal, Canada, 2021). All eggs were segmented by: (1) enforcing contrast thresholds to isolate the fossils from the matrix (Fig. 4.2–4.3); (2) creating regions of interest; (3) and applying false colors for segmentation (Fig. 4.4). Backscatter Electron image (BSE) and Energy-dispersive X-ray spectroscopy (EDS) was conducted on a single isolated egg (Egg One) using a FEI Quanta 200 FEG Environmental SEM, with an EDAX Octane Plus SDD x-ray detector in Low Vacuum mode with chamber pressure of 70 Pa to analyze the surface composition of Egg One at 80x magnification with a scanning energy of 12 kv at the University of Windsor, Toronto, Canada. The single egg had initially been glued using cyanoacrylate on a toothpick for analysis at the synchrotron. Some residues of cyanoacrylate could not be removed, explaining local concentrations of carbon.