sample preparation for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope

<meta charset="utf8"></head><body>ゼブラフィッシュ胚のための洗練されたマルチビューライトシート顕微鏡法

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 requirement1,2,3,4,5,6,7. 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 ends, illuminating only the plane of interest. A detection objective is placed orthogonally, and then the emitted fluorescent light from all illuminated points in the sample is collected simultaneously. A 3D image is then obtained by sequentially moving the embryo through the static light sheet.
In addition, in a specific form of this methodology, termed multiview light sheet microscopy, the samples can be suspended in a polymer tube that can be rotated using a rotor, enabling imaging of the same embryo from multiple angles9,10,11. Following imaging, the images from multiple angles are fused based on registration markers, which are typically globular fluorescent markers within the embryo (e.g., nuclei) or in the tube (e.g., fluorescent beads). Multiview imaging and fusion significantly improve the axial resolution, providing isotropic resolution across all three dimensions12. While this is a big advantage, a major challenge of multiview methodology is sample mounting, where embryos have to be mounted and kept in place in the tubes during the entire time course of imaging.
For performing multiview imaging, to keep the embryos in place and prevent movement while imaging, embryos can be embedded in agarose. However, this often leads to detrimental growth and development, particularly for early-stage zebrafish embryos13, the model system that is discussed here. A second mounting strategy is to use a thin tube that is only slightly bigger than the diameter of the embryo, where the embryo can be pulled into the tube along with the embryo medium, followed by closing the bottom of the tube with an agarose plug14. In this method, because the tube is filled with embryo medium, registration markers such as fluorescent beads cannot be used for the fusion of the different views, and registration is therefore reliant on markers within the embryo. In general, beads act as better registration markers as the signal of the markers within the embryo degrades upon moving deeper into the sample owing to both illumination and detection limitations of any microscope.
Thus, a third approach, which will be detailed here and used previously5,13,14,15,16, is imaging early zebrafish embryos with an intact chorion and filling the tube with a minimal percentage of agarose, which contains beads as registration markers. In this scenario, because manual intervention for positioning embryos within a chorion is not possible, this study provides statistics on the default orientation early zebrafish embryos fall into, particularly focusing on 70% epiboly and bud stages. It then discusses the optimal number of views required for imaging early-stage embryos at cellular-scale resolution and details the process of fusion using BigStitcher, a FIJI-based plugin10,17,18. Together, this protocol, which uses a 20x/1 NA objective, aims to facilitate zebrafish embryologists in using multiview light sheet systems for imaging embryos with nuclei and membrane markers from gastrulation to early somite stages.

<meta charset="utf8"></head><body>著者スポットライト:高解像度マルチビューライトシート顕微鏡のためのゼブラフィッシュ胚のマウント戦略 — イメージングと画像再構成の技術

初期ゼブラフィッシュ胚のライトシート顕微鏡イメージングとマウント戦略

ライトシート顕微鏡を用いた無傷の絨毛膜内の初期ゼブラフィッシュ胚のイメージングのためのサンプル調製

sample-preparation-for-imaging-early-zebrafish-embryos-within-an

Research

JoVE Journal

Developmental Biology

Sample Preparation for Imaging Early Zebrafish Embryos Within an Intact Chorion Using a Light-Sheet Microscope

54 ビュー. まず、ビードを加えた準備したアガロースチューブを摂氏37度の水浴から取り出します。10ミリリットルのアガロース全体を6センチのペトリ皿に注ぎます。温度が摂氏33度を下回ったら、10〜15個の胚をアガロース溶液に移します。ペトリ皿を渦巻かせて、転送されるはずの小さなバッファーが十分に分散されるようにします。次に、適切な?...

ビデオ: ライトシート顕微鏡を用いた無傷の絨毛膜内の初期ゼブラフィッシュ胚のイメージングのためのサンプル調製

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

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.

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.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...