一种用于斑马鱼胚胎长时间延时共聚焦显微镜观察的分层固定方法。
A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos.
作者信息
Upadhyay Sanat, Vergara Leoncio, Shah Pranjali, Gustafsson Jan-Åke, Kakadiaris Ioannis, Bondesson Maria
机构信息
Computational Biomedicine Lab, Texas Institute of Measurement Evaluation and Statistics, University of Houston.
Institute of Biosciences and Technology, Texas A&M Health Science Center.
出版信息
J Vis Exp. 2020 Jan 14(155). doi: 10.3791/60321.
Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.
发育动力学可以通过对在特定组织或细胞中表达荧光的活转基因斑马鱼胚胎进行共聚焦延时显微镜观察来跟踪。对整个胚胎发育进行成像的一个困难在于斑马鱼胚胎的长度会大幅增长。当像常规那样将其固定在0.3 - 1%的低熔点琼脂糖中时，琼脂糖会限制生长，导致柔软的胚胎身体变形。然而，为了进行共聚焦延时显微镜观察，胚胎必须被固定。本文描述了一种用于斑马鱼胚胎的分层固定方法，该方法在限制胚胎运动的同时允许其不受限制地生长。固定是在不同浓度的琼脂糖层中进行的。为了证明该方法的可用性，对转基因鱼的整个胚胎血管、神经元和肌肉发育进行了连续55小时的成像。这种固定方法可用于使用倒置显微镜对整个斑马鱼胚胎进行简便、低成本的成像，无需模具或特殊设备。
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