Tetrahedral serial multiview microscopy and image fusion for improved resolution and extent in stained zebrafish embryos

Background: Spatial mapping on the single-cell level over the whole organism can uncover roles of molecular players involved in vertebrate development. Custom microscopes have been developed that use multiple objectives to view a sample from multiple views at the same time. Such multiview imaging approaches can improve resolution and uniformity of image quality as well as allow whole embryos to be imaged (Swoger et al., Opt Express, 2007;15(13):8029). However, multiview imaging is highly restricted to specialized equipment requiring multiple objectives or sample rotation with automated hardware. Results: Our approach uses a standard single-objective confocal microscope to perform serial multiview imaging. Multiple views are imaged sequentially by mounting the fixed sample in an agarose tetrahedron that is manually rotated in between imaging each face. Computational image fusion allows for a joint 3D image to be created from multiple tiled Z-stacks acquired from different angles. The resulting fused image has improved resolution and imaging extent. Conclusion: With this technique, multiview imaging can be performed on a variety of common single-objective microscopes to allow for whole-embryo, high-resolution imaging.