Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.
Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.
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DOI：
10.3791/53966
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发表时间：
2016-04-10
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影响因子：
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通讯作者：
Norden C
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文献类型：
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作者：
Icha J;Schmied C;Sidhaye J;Tomancak P;Preibisch S;Norden C
光片荧光显微镜（LSFM）作为一种成像胚胎发育的方法越来越受欢迎。与共聚焦系统相比，LSFM的主要优点是其低光毒性，温和的安装策略，高信噪比的快速采集以及从各种角度（视图）长时间成像样品的可能性。从多个视图成像释放了LSFM的全部潜力，但同时它可以创建TB大小的数据集。处理这样的数据集是使用LSFM的最大挑战。在本协议中，我们概述了这个问题的一些解决方案。直到最近，LSFM主要是在拥有专业知识的实验室中进行，以建立和操作自己的光片显微镜。然而，在过去的三年里，LSFM的几个商业实现变得可用，这是多用途的，易于使用的任何发育生物学家。本文主要针对那些研究人员，谁不是LSFM技术开发人员，但希望采用LSFM作为一种工具来回答特定的发育生物学问题。 在这里，我们使用斑马鱼眼睛发育成像作为一个例子，向读者介绍LSFM技术，我们展示了LSFM在多个空间和时间尺度上的应用。本文介绍了一个完整的实验方案开始安装斑马鱼胚胎LSFM。然后，我们概述了使用市售光片显微镜成像的选项。重要的是，我们还解释了使用作为斐济插件实现的开源解决方案进行多视图数据集后续注册和融合的管道。虽然该协议的重点是成像发育中的斑马鱼眼睛和处理数据从一个特定的成像设置，大多数的见解和故障排除建议在这里是通用的，该协议可以适应各种光片显微镜实验。
Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.