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. 2021 Feb 11;10:e61408. doi: 10.7554/eLife.61408

Figure 6. 3D whole-brain nuclei detection pipeline.

(A) Overview of the nuclei detection and assignment pipeline. After tissue clearing by CUBIC and nuclear staining, we imaged intact whole P0 mouse brains by lightsheet microscopy (Figure 6—figure supplement 1). The E18.5 mouse brain atlas was registered to volumetric sample images. Nuclei were identified brain-wide with a 3D blob detector algorithm, and the nuclei coordinates were used to map to specific region labels, allowing quantification of nuclei counts and density for each label. (B) Axial view of the hippocampus of a P0 mouse brain at the original magnification of 5x (left) and zoomed to 20x (right), with the same region of interest (ROI) depicted by the yellow box in both. (C) Serial 2D planes along the ROI shown by the yellow box in part ‘B’ show the emergence and disappearance of nuclei while progressing vertically (z-axis) through the ROI. Each of four individual nuclei is assigned a unique colored circle. The most central image of the nuclei is indicated by a solid circle and asterisk, while images above or below have dashed circles. (D) 3D schematic of the four nuclei from part ‘C’ demonstrating their spatial orientation.

Figure 6.

Figure 6—figure supplement 1. Custom 3D printed specimen mount for suspending samples in the Zeiss Lightsheeet Z.1 microscope.

Figure 6—figure supplement 1.

(A) Mount schematic and orientation in the chamber. The lightsheet chamber requires the specimen to be suspended between the two illumination objectives producing a plane of light and detection objective perpendicular to this plane. To hold the brain in place while immersed in the refractive index matching solution, we devised a mount with a platform to maximize surface contact with the ventral surface of the brain, attached by super glue. To allow full latitude of the specimen within the chamber, we added a mount neck to center the specimen between the illumination objectives when the manipulator was approximately centered. (B) Photograph of mount and custom mount holder to facilitate gluing the specimen to the mount platform. Cotton buds were used to dry the ventral surface of the brain before attachment. We used the rod end of another mount to gently press the brain into the glue. (C) Photograph of specimen suspended and centered between the illumination objections, facing the detection objective.
Figure 6—figure supplement 2. Nuclei detection optimization and comparison with intensity.

Figure 6—figure supplement 2.

(A) Example receiver operating characteristic (ROC) curves. (top) Example ROC of nuclei detection across varying isotropic scaling factors along the z-axis. Scaling is shown as the fraction of isotropy along the z-axis, where 1 = isotropic. For example, 0.5 indicates that each ROI was resized so that the z-axis was half of the size it would need to be for full isotropy with the x- and y-axes. (bottom) Example ROC evaluating two hyperparameters, the minimum and maximum standard deviations for the Gaussian kernel used for multi-scale detection, to find the optimal size bounds for blob detection. Inset shows a zoomed view of the clustered points. (B) Total nuclei vs. intensity by label showed a linear relationship using labels defined by either the original (mirrored; r = 0.997, p≤1×10−16) or smoothed (edge-aware; r = 0.997, p≤1×10−16) atlases. In the correlation plot (bottom), the size of the circle indicates the size of the correlation coefficient (r). (C) Similarly, nuclei density vs. intensity density relationships were approximately linear in the original (mirrored; r = 0.894, p≤1×10−16) and smoothed (edge-aware; r = 0.929, p≤1×10−16) atlases. (D) Sensitivity and False Discovery Rate stratified by nuclei density in manually annotated truth sets as seen in the histogram (left) showed a similar distribution by region (middle) and overall metrics (right) by density group, with numerically lower recall and slightly higher precision in denser regions.
Figure 6—figure supplement 3. Registration of atlas to WT brains.

Figure 6—figure supplement 3.

(A) Overlays of each registered atlas microscopy image on its corresponding downsampled wild-type brain. All examples are shown at the same plane. (B) Composite image of wild-type sample brains registered in reverse, using the same settings but registering the sample to the atlas to evaluate overall alignment of registered brains. (C) Overall registration quality as expressed by the DSC between the foreground of each sample and its registered atlas showed a median of 0.91 (mean 0.90).