A range of sample-to-scintillator distances (SSD) was surveyed for phase contrast enhancement of edges in larval samples. A line profile (A) is drawn through the retinal region as denoted by the yellow line in (B) to highlight differences in voxel intensity at edges. Zoomed insets (C) show the photoreceptor layer aligned with the attenuation profile in (A). The attenuation range (maximum attenuation– minimum attenuation) is shown in (D) for homogenous (lens, inner plexiform layer) and variable (photoreceptor layer) regions. The attenuation range increases for variable regions but remains constant for homogenous regions as SSD increases, demonstrating the effects of phase contrast edge enhancement. A SSD of 30 mm provided a level of edge perception resembling that found in traditional glass slide histology and was used for all subsequent acquisitions.

A juvenile (33 dpf) zebrafish was scanned using pink-beam (A) and monochromatic (B) sources. Insets show the ability for pink-beam (C) and monochromatic beam (D) to resolve the fine detail found in zebrafish photoreceptor layer. Signal-to-noise ratio (SNR) in high (eye lens) and low attenuation (brain) tissues was compared for both images (E). The monochromatic beam image has a higher SNR than the pink-beam image in both cases. While the noise in the pink beam image could be reduced by increasing acquisition times, the contrast in the monochromatic image is inherently better. A line profile (F) through the photoreceptor layer shows that the monochromatic beam is superior for discerning edges, which can be attributed to the phase contrast optimization done in Figure 1—figure supplement 1. Under these pink-beam imaging conditions, phase-contrast enhancement, which is energy-dependent, gets averaged out.

Coronal (A, H), sagittal (B, I), and axial (C–F, J–M) cross sections of 4 dpf larval (A–G) and 33-dpf juvenile (H–N) wild-type zebrafish acquired using synchrotron X-ray micro-tomography at 0.743 μm³ and 1.43 μm³ isotropic voxel resolution, respectively. 3D volume renderings (G, N) show the cross-sections in relation to the whole organism. In contrast to histology, the cross sections are a single voxel in thickness and can be obtained in any plane (including oblique cuts) after imaging. Complete cross-sections in the orthogonal directions for both fish are provided (Videos 2 and 3). Images are presented to match histological convention of dark cell nuclei (higher attenuation is darker).

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).

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).

A 3D rendering of a whole juvenile (33 dpf) zebrafish is presented with highlighted gastrointestinal (GI) tract (A). The GI tract exemplifies a convoluted structure for that can be unraveled for histological analysis. The isolated GI tract is displayed (B). Isotropic resolution of data permits virtual slicing at any angle (B1–B3) without a decrease in resolution, allowing comprehensive histology-like visualization despite its tortuous nature. A spline-based reslicing method for visualizing serpentine organs across their entire path is also shown (C). A nonlinear cutting plane (blue) follows the structure of interest, allowing the cut to render a structure’s total length onto a single plane (D).

Orthogonal views (middle), a zoomed view (top), and a projection zoom view (bottom, 36 slice, 50 µm projection) of the head of a juvenile (33 dpf) zebrafish are presented. Sagittal, coronal, and transverse sections are denoted by red, yellow, and blue lines, respectively.

Orthogonal views (middle), a zoomed view (top), and a projection zoom view (bottom, 36 slice, 50 µm projection) of the head of a juvenile (33 dpf) zebrafish are presented. Sagittal, coronal, and transverse sections are denoted by red, yellow, and blue lines, respectively.

Top: Cutout visualization of both wild-type and huli hutu larval (five dpf) zebrafish stained with PTA showing detail in many soft tissue structures. Bottom: Cell types and structures that can be visualized include neuronal cells in the eye (A), cartilaginous rudiments of the squamous patch on the dorsal (arrow) pharynx (B), nucleated red blood cells (C), intact pneumatic duct (* to arrow) and goblet cells in the gut (D), and cross-striations of bands of muscles encircling the swim bladder (E). Panels A and D represent individual slices (0.743 μm in thickness) while B, C, E represent maximum intensity projections of 5 μm thick sections to visualize larger 3D structures. Compared to age matched wild-type larval zebrafish (top), the number of neuronal cells in the eye are markedly reduced (A’), chondrocytes appear cytologically normal, but formation of cartilaginous structures is markedly reduced (B’), the myocardium is thickened and, as is evident from a survey through all the sections of the heart (a single slice shown here) contains a markedly reduced number of nucleated red blood cells, consistent with anemia and abnormal hematopoiesis (C’). We are able determine the absence of the pneumatic duct and swim bladder in hht due to the ability to scan through the full volume of the sample. D’ shows degenerate tissue and E’ other tissues where those organs normally lie. A’, B’ and D’ represent individual slices (0.743 μm in thickness) while C’ and E’ represent maximum intensity projections of 5 μm thick sections to visualize structures of larger dimension.

75 × 75×75 µm regions were selected on each fish to validate detection of nuclei: sample regions R1, R2 and R3 are presented (A). 3D visualizations of thresholded original data (B–a) and nuclear probabilities based on the trained classifier (B–b) in Region two are shown. 3D rendering of validation is presented in (B–c); green denotes true positive, blue false positive, orange false negative. Good agreement between manual and automated segmentation is shown on regional 3D rendering and f1 scores (C) across all specimens.

75 × 75×75 µm regions were selected on each fish to validate detection of nuclei: sample regions R1, R2 and R3 are presented (A). 3D visualizations of thresholded original data (B–a) and nuclear probabilities based on the trained classifier (B–b) in Region two are shown. 3D rendering of validation is presented in (B–c); green denotes true positive, blue false positive, orange false negative. Good agreement between manual and automated segmentation is shown on regional 3D rendering and f1 scores (C) across all specimens.

PTA and osmium staining in (A) micro-CT and (B) transmission electron microscopy images (Hildebrand et al., 2017) both allow the nuclei of individual cells to be distinguished. Automated object detection applied to these images show similar regional patterning and counts of nuclei in corresponding brain slices (C, D).

Shape and volume of manually detected brain cell nuclei varies between red blood cells (RBCs) and motor neurons (n = 20 cells each) (A, B). The mean elongation was 1.9, 1.3, and 1.1 for RBCs, motor neurons, and brain nuclei, respectively. The mean volume was 99, 102, and 13 µm³ for RBCs, motor neurons, and brain nuclei, respectively. These releative values are consistent with what is visually apparent in both histology (C1 and C3) and histotomography (C2 and C4). Volumes of motor neuron and erythrocytes include both nuclei and cytoplasm. Differences in the distribution of brain nuclei were also observed. Cell nuclei were also identified across whole zebrafish samples via a manually trained classifier and segregated by registering brain regions for five five dpf samples (D). The regions identified through anatomical landmarks include olfactory epithelium, telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon, hypothalamus, spinal cord, and white matter. The proportion of cells per brain region as percentage of total cell counts, agree in rank order between samples (E). Computed elongation and volumes showed no significant difference between individual fish (F, G).

Cellular density varies between individual brain regions and carries distinct signals consistent between individual samples (A). 3D renderings of whole-brain densities with identical transparency settings are presented for each fish (B) and reflects signal differences presented in (A).