Standardized mounting method of (zebrafish) embryos using a 3D-printed stamp for high-content, semi-automated confocal imaging

David Simon Kleinhans,Virginie Lecaudey

doi:10.1186/s12896-019-0558-y

Abstract

BackgroundDevelopmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be easily observed and imaged using fluorescent proteins have become very popular. One limiting factor however is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. One way to improve this is by developing mounting methods to increase the number of embryos that can be imaged simultaneously in near-to-identical orientation.ResultsHere we present an improved mounting method allowing semi-automated and high-content imaging of zebrafish embryos. It is based on a 3D-printed stamp which is used to create a 2D coordinate system of multiple μ-wells in an agarose cast. Each μ-well models a negative of the average zebrafish embryo morphology between 22 and 96 h-post-fertilization. Due to this standardized and reproducible arrangement, it is possible to define a custom well plate in the respective imaging software that allows for a semi-automated imaging process. Furthermore, the improvement in Z-orientation significantly reduces post-processing and improves comparability of volumetric data while reducing light exposure and thus photo-bleaching and photo-toxicity, and improving signal-to-noise ratio (SNR).ConclusionsWe present here a new method that allows to standardize and improve mounting and imaging of embryos. The 3D-printed stamp creates a 2D coordinate system of μ-wells in an agarose cast thus standardizing specimen mounting and allowing high-content imaging of up to 44 live or mounted zebrafish embryos simultaneously in a semi-automated, well-plate like manner on inverted confocal microscopes. In summary, image data quality and acquisition efficiency (amount of data per time) are significantly improved. The latter might also be crucial when using the services of a microscopy facility.

Highlights

Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes
The protocol we describe here was designed to be used with XY scanning universal sample holders that usually come with any motorized-stage inverted microscope
The lateral line comprises hundreds of NMs distributed over the entire body surface

Summary

Introduction

Developmental biology relies to a large extent on the observation and comparison of phenotypic traits through time using high resolution microscopes. In this context, transparent model organisms such as the zebrafish Danio rerio in which developing tissues and organs can be observed and imaged using fluorescent proteins have become very popular. One limiting factor is the acquisition of a sufficient amount of data, in standardized and reproducible conditions, to allow robust quantitative analysis. To process biological samples of whole organisms in a high-content manner, it is important to have a standardized way of sample mounting, data acquisition, data processing and analysis. High-resolution imaging of multiple samples in standardized conditions is of key importance

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