Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development

Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.

Organs and subdomain segmentation. Organ segmentation was performed using the segmentation tools of Amira software (FEI Visualization Sciences Group, USA). The borders of the region were highlighted in sections of the three-dimensional reconstructed volume. This was performed using a feature of the software that forces the highlighted border to lie on an automatically detected edge. The borders were manually highlighted in three to five sections, along three perpendicular directions. The software then allowed the user to automatically create a three-dimensional segmentation volume that included the highlighted borders and interpolated the volume using a gradient-based detection.

Visualization.
Sections and 3D volumes were visualized using Fiji (Schindelin et al., 2012). In order to generate weighted intensity projections ( Fig. 2 and supplementary materials Fig. S3) of a 3D data stack, each image of the stack was first multiplied by a weighting factor. The factor varied linearly from 1 (first slice) to 0.5 (last slice). Then, a maximum intensity projection was performed. Compared to simple maximum intensity projection this method provided more details on one side of the sample and was useful to visualize the obtained results from different angles.    All images of the time-lapse reconstructions were registered to a reference system based on the fish's anatomy. The position of the eye lenses was automatically detected in the reconstructed volumes. A reference system with origin in the eyes' center of mass was defined and a rigid transformation was applied to the reconstructed volumes in order to orient the eyes of the zebrafish to the same virtual axis (horizontal) in all the time points of the time lapse (see also Movie 6). The fluorescence volumes obtained with SPIM were aligned accordingly, applying the same rigid transformation.

Figure S7 | Sample holder
The sample holder was constructed using a precision xy translation mount (Thorlabs, LM1XY). The two translation screws (blue arrows) were used to move the sample relative to the rotational axis of the rotating stage of the microscope (not shown) and to center the sample in the field of view during the entire 360° rotation. The translation mount was modified to incorporate three magnets to mount the sample holder on the rotating stage (green arrows). The sample (red arrow) is embedded in agarose inside a glass capillary and extruded for imaging. a b c d e

Movie 1 | Spiral acquisition animation and raw data
During acquisition the sample is continuously translated and rotated across the image plane of the detection objective. The specimen is sampled along a spiral. The raw data acquired during the sample movement are shown on the right hand side.

Movie 2 | Tomographic reconstruction
Sagittal (left side), coronal (center) and transverse (right side) sections through a 4 dpf zebrafish (head and trunk regions) reconstructed with optical tomography. Scale bar is 100 µm.

Movie 3 | Segmented organs
Three-dimensional reconstruction of the segmented organs of a wild-type 2 dpf zebrafish head (also shown in Fig. 1 and Fig. S1). The organs are: retina (pink), eye lens (red), brain ventricles (green), brain (cyan), olfactory pit (yellow), epiphysis (blue). The raw reconstructed data are superimposed to the segmented organs, around half of the sample.