Figure 3. Five micron thick maximum intensity projections of synchrotron X-ray histotomographic images resemble histology.

Top: Comparison of larval (five dpf) zebrafish obtained from a 5 μm thick histology section and micro-tomography data (A and B, respectively). The larval micro-tomograph is 0.743 μm³ in isotropic voxel resolution, and a maximum intensity projection (MIP) of 7 slices, totalling 5.20 μm, resembling the appearance of ~5 μm thick histological sections. Bottom: Comparison of juvenile (33 dpf) zebrafish i5 μm histology section and micro-tomographic MIPs (C and D, respectively). Juvenile scan data is of 1.43 μm³ isotropic voxel resolution, and a MIP of 3 slices, totalling 4.29 μm thickness, is shown. Insets show detail of brain cell nuclei (A–B) and delicate gill structure (C–D). The images demonstrate the near histological resolution of X-ray histotomography. While natural variation in the size of specific features is observed in age matched fish (panel C length = 7.8 mm, panel D length = 10 mm) individual histological features are consistent.

Figure 3—figure supplement 1. Bands of skeletal muscle wrapping the air bladder of larval zebrafish allow characterization of resolution.

The biological validation of imaging resolution was performed through measurement of distances between Z-lines in the swim bladder striated muscle strands. Cross sections of striated muscle surrounding the swim bladder diagonally in larval (five dpf) zebrafish (A) was used to generate pixel intensity profiles (B). Measurement of pixel distances between local intensity maxima assuming 0.743 μm³ isotropic voxel resolution yielded an average sarcomere length of 2.16 μm (SD = 0.55 μm for 293 distance measurements), consistent with published sarcomere lengths in larval zebrafish (Burghardt et al., 2016; Dou et al., 2008).