Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Summary

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.

Abstract

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Introduction

Ensuring minimal phototoxicity is a major requirement for imaging live embryos with high spatiotemporal resolution for long periods. Over the last decade, light sheet microscopy has become the methodology of choice to meet this requirement^{1,2,3,4,5,6,7}. Briefly, in this technique that was first used in 2004 to capture developmental processes⁸, two aligned thin sheets of laser pass through the embryo from opposing e....

Protocol

The zebrafish maintenance and experimental procedures used in this study were approved by the institutional animal ethics committee, vide Reference TIFR/IAEC/2023-1 and TIFR/IAEC/2023-5. Embryos obtained by crossing heterozygous fish expressing Tg(actb2:GFP-Hsa.UTRN)¹⁹ were injected with H2A-mCherry mRNA (30 pg) at the one-cell stage. H2A-mCherry mRNA was synthesized using the pCS2+ H2A-mCherry plasmid (a gift from the Oates lab, EPFL) by in vitro transcription. Embryos expr.......

Discussion

Positioning an embryo in the right orientation to image the region of interest is one of the rate-limiting steps that often results in a failed microscopy session for a user. This is more so in a multiview light sheet microscope where manual manipulation of the orientation is difficult as the samples are embedded within a tube. To aid in this process, this study reports the statistics of various positions a zebrafish embryo takes up between 70% epiboly and early somite stages within a chorion when the polymer tube with e.......

Materials

Name	Company	Catalog Number	Comments
Agarose, low gelling temperature	Sigma-Aldrich	A9414
Calcium Chloride dihydrate	Sigma-Aldrich	12022
FIJI			Version: ImageJ 1.54f
Latex beads, carboxylate-modified polystyrene, fluorescent red, 0.5 μm mean particle size, aqueous suspension	Sigma-Aldrich	L3280
Magnesium sulfate heptahydrate	Sigma-Aldrich	M2773
mMESSAGE mMACHINE SP6 Transcription kit	ThermoFischer Scientific	AM1340	For in vitro transccription of H2A-mCherry plasmid
Potassium Chhloride	Sigma-Aldrich	P9541
Potassium phosphate monobasic	Sigma-Aldrich	P0662
PTFE Sleeving AWG 15L - 1.58 mm ID x 0.15 mm Wall +/-0.05	Adtech Innovations in Fluoroplastics	STW15	PTFE tubes
Sodium Chloride	Sigma-Aldrich	S3014
Sodium phosphate dibasic	Sigma-Aldrich	71640
Ultrasonic Cleaner	Labman	LMUC3	Ultrasonicator
Zeiss LightSheet 7 System	Zeiss