Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope

Area of Science:

• Developmental Biology
• Microscopy Techniques
• Zebrafish Embryology

Background:

• Light sheet microscopy, including selective plane illumination microscopy (SPIM), is valuable for long-term, time-lapse imaging of biological development.
• Current sample mounting methods, like embedding zebrafish embryos in agarose, often impede development and yield unreliable data.
• Fragile sample mounting remains a significant limitation for advanced microscopy applications.

Purpose of the Study:

• To systematically evaluate and optimize sample preparation for light sheet microscopy of zebrafish embryos.
• To quantify the viability and mobility of zebrafish embryos under novel mounting conditions.
• To provide guidelines for anesthetic use in zebrafish embryo imaging.

Main Methods:

• Systematic quantification of zebrafish embryo viability and mobility.
• Development and testing of sample mounting techniques using fluorinated ethylene propylene (FEP) tubes with varying agarose and methylcellulose concentrations.
• Comparison of different Tricaine concentrations on zebrafish development.

Main Results:

• Fluorinated ethylene propylene (FEP) tubes with low-concentration agarose or methylcellulose offer optimal embryo confinement and optical clarity for 3-day imaging.
• Established optimal conditions for mounting fragile zebrafish embryos for light sheet microscopy.
• Provided guidelines for Tricaine use in zebrafish embryo studies.

Conclusions:

• Optimized sample preparation techniques enhance the applicability of light sheet microscopy for long-term zebrafish embryo imaging.
• Improved mounting methods support in toto and in vivo imaging, benefiting techniques like optical projection tomography (OPT).
• This research advances developmental biology by enabling more reliable and extensive live imaging of embryos.