Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Despite internal asymmetries along the left-right axis, in most higher organisms, external structures such as skeleton and muscle develop in a symmetric manner. Using zebrafish embryos, we combine high-resolution live imaging, quantitative analysis, and theoretical modeling to investigate the principles of symmetry establishment. The field heavily depends on various microscopy techniques for imaging developing embryos with high spatiotemporal resolution, followed by quantitative analysis of the acquired images.

Different forms of microscopy provide unique advantages. However, of late, light-sheet and live super-resolution microscopy have significantly advanced our view of dynamic processes in embryonic development. Traditional confocal and multiphoton microscopy often lead to photobleaching and phototoxicity, in addition to a limited field of view when imaging live embryos.

A multiview light-sheet microscope addresses these issues, allowing for longer observation times and sample rotation. However, challenges remain in sample preparation and mounting for imaging using this technique. A major limitation of a successful imaging session is mounting and orienting the sample appropriately.

Here we describe strategies to mount early zebrafish embryos for imaging in a multiview light-sheet microscope and the general procedure for acquiring and reconstructing multiview images.