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

<meta charset="utf8"></head><body>Protocollo di microscopia a foglio luminoso multiview perfezionato per embrioni di zebrafish

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>Autore in primo piano: Strategie per il montaggio di embrioni di zebrafish per la microscopia a foglio luminoso multivista ad alta risoluzione - Tecniche per l'ima

Imaging al microscopio a foglio leggero e strategie di montaggio per embrioni precoci di zebrafish

Preparazione del campione per l'imaging di embrioni precoci di zebrafish all'interno di un corion intatto utilizzando un microscopio a foglio luminoso

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

53 Views. Per iniziare, rimuovere il tubo di agarosio preparato con l'aggiunta di perline da un bagno d'acqua a 37 gradi Celsius. Versare tutti i 10 millilitri di agarosio su una capsula di Petri da sei centimetri. Una volta che la temperatura scende sotto i 33 gradi Celsius, trasferisci da 10 a 15 embrioni nella soluzione di agarosio.Agitare la capsula di Petri in modo che il piccolo buffer che sarebbe stato trasferito si disperda bene. Quindi, prendere una mic...

Video: Preparazione del campione per l'imaging di embrioni precoci di zebrafish all'interno di un corion intatto utilizzando un microscopio a foglio luminoso

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 ...