Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos
July 19th, 2024
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.
Overview
This article describes a sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope. It focuses on the orientations of embryos at the 70% epiboly and bud stages and outlines imaging strategies for achieving cellular-scale resolution.
Key Study Components
Area of Science
- Neuroscience
- Developmental Biology
- Imaging Techniques
Background
- Higher organisms often develop external structures symmetrically despite internal asymmetries.
- Zebrafish embryos serve as a model for studying symmetry establishment in development.
- High-resolution live imaging is crucial for understanding dynamic processes in embryonic development.
- Various microscopy techniques have unique advantages and limitations.
Purpose of Study
- To investigate the principles of symmetry establishment in zebrafish embryos.
- To utilize advanced imaging techniques for better visualization of embryonic development.
- To analyze the orientations of embryos within the chorion during critical developmental stages.
Methods Used
- Light-sheet microscopy for imaging embryos.
- Quantitative analysis of acquired images.
- Theoretical modeling to understand symmetry establishment.
- Comparison of different microscopy techniques.
Main Results
- Light-sheet microscopy provides cellular-scale resolution of zebrafish embryos.
- Embryos exhibit different orientations within the chorion at the analyzed stages.
- Advanced imaging techniques reduce issues like photobleaching and phototoxicity.
- Insights into dynamic processes of embryonic development were gained.
Conclusions
- The study enhances understanding of symmetry in embryonic development.
- Light-sheet microscopy is a valuable tool for live imaging of embryos.
- Future research can build on these findings to explore further developmental processes.
Frequently Asked Questions
Chapters in this video
0:00
Introduction
1:37
Sample Preparation for Imaging Early Zebrafish Embryos Within an Intact Chorion Using a Light‐Sheet Microscope
2:53
Analyzing Different Orientations of Zebrafish Embryos Within the Chorion Using Multiview Imaging
4:53
Multiview Image Analysis to Obtain Cellular-Scale Resolution Throughout the Embryo
