X tek hmxst 225

SAM-PK-5867 and SAM-PK-6047A were scanned using X-ray CT at the Evolutionary Studies Institute, University of the Witwatersrand, Johannesburg, South Africa. Scanning was undertaken with an X Tek HMX ST 225 (Nikon Metrology Inc.), comprising 3000 projections, using a tungsten target with gain 4. Files were reconstructed using CT Pro 3D software (Nikon Metrology, Inc.). Scan settings were as follows: SAM-PK-5867—70 kV, 140 µA, 1000 ms, 57.50 µm voxel size, 1.8 mm Al filter; SAM-PK-6047A—120 kV, 95 µA, 2000 ms, 60.10 µm voxel size, 1.2 mm Cu filter. UMZC T.692 was CT scanned at the Natural History Museum, London, UK, with an X Tek HMX ST 225 (Nikon Metrology Inc.), comprising 3142 projections, using a tungsten target with gain 4. Scan settings were as follows: 210 kV, 250 µA, 500 ms, 98.8 µm voxel size, 2.5 mm Cu filter. All scans were post-processed, segmented and examined using VG Studio Max 2.1 and 2.2, and myVGL 3.3.2 (Volume Graphics, Heidelberg, Germany). The scans of UMZC T.692 are archived at the Zenodo repository (doi:10.5281/zenodo.3887056), and those of other specimens will be archived following completion of work by B.-A.S.B. and are available upon request. Surface renderings of all CT-scanned specimens have also been made available as mesh files at Zenodo (doi:10.5281/zenodo.3887056).

We subjected our F₅ hybrid population to x‐ray micro computed‐tomography (µCT) scanning to obtain 3D information on the neurocranium and brain shape. All hybrids were scanned twice: first to obtain hard‐tissue morphology (i.e., skeletal tissues), and second to gain soft‐tissue morphology following contrast‐enhanced staining protocols (i.e., neural tissues). We performed all µCT scanning using an X‐Tek HMXST 225 (Nikon Corporation). Hard‐tissue skeletal scans were acquired at 25–35 µm resolution using 95 kV and 90 µA. We captured soft neural tissue morphology by submerging our hybrid cichlids in 2.5% Lugol's iodine solution for 24 h. Lugol's iodine is radiopaque, permitting the visualization of the brain via µCT scanning once the solution has fully penetrated neural tissues (Hedrick et al., 2018 (link)). All iodine‐stained specimens were scanned at 20–25 µm resolution at 115 kV and 105 µA with a 0.1 mm copper filter. We then segmented the hard and soft tissues using Mimics (v.19 Materialise NV) and exported the 3D models to Geomagic (v.1.0 3D Systems) to remove noise (i.e., small floating voxels) by filtering for small, disconnected components before morphometric analysis.

Before mechanical testing all 30 of the tensile trabecular sections were imaged using a micro-CT system (Nikon X-Tek HMXST-225: tungsten target; X-Ray beam 180 keV and 200 µA, voxel size 30 µm; 3,124 projections; 360° rotation). Total scan time was 30 min. Scans were used to calculate the bone volume fraction (BV/TV) for the entire tissue section (not just the region which would be imaged using scattering and diffraction). Scans contained 256 grey values and were segmented into binary images using the grey value frequency distribution plot to apply a global threshold at the minima between the two peaks that represented marrow and bone^{42 –44} . The advantage of this method is that variations in bone density or scan performance are taken into account and do not affect structural measurements (e.g. BV/TV). The minima at which the threshold was placed ranged between 190 and 233 for the Controls, 200–225 for the Fx-Untreated and 206–222 for Fx-BisTreated. All of the voxels above the threshold were defined as bone and the values below as background. Bone volume fraction was calculated from the binary images using the BoneJ plugin for ImageJ (v1.49) by dividing the total number of voxels that represented bone by the total number of voxels in the whole scan volume.