Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.
Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.
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DOI：
10.1186/s12861-016-0122-0
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发表时间：
2016-06-30
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通讯作者：
Telford MJ
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作者：
Girstmair J;Zakrzewski A;Lapraz F;Handberg-Thorsager M;Tomancak P;Pitrone PG;Simpson F;Telford MJ
选择性平面照明显微镜（SPIM，一种光片显微镜）涉及聚焦一薄层激光通过一个样本在直角的物镜透镜。由于仅照射透镜的焦平面处的样品的薄部分，因此自然不存在焦点外的光，并且由于光的毒性（光毒性）大大降低，使得能够进行更长时间的实时成像。OpenSPIM是一个开放式访问平台（Pitrone等人，2013年和OpenSPIM.org），旨在为新用户提供构建SPIM显微镜基本配置的分步说明，原则上可以根据每个实验室自己的要求和预算进行调整和升级。在这里，我们描述了我们自己的经验，设计，构建，配置和使用OpenSPIM的过程中，我们的研究到早期发展的polyclad扁虫Maritigrella crozieri -一个非模型动物。我们的OpenSPIM基于标准设计，增加了双色激光照明，可同时检测两种探针/分子和双面照明，从而在整个样品中提供更均匀的信号强度。我们的OpenSPIM提供高分辨率3D图像和延时记录，我们演示了双色激光的使用和双色双面成像的好处。我们用显微镜研究了多包层扁虫M. crozieri我们的显微镜的能力证明了我们的能力，记录定型螺旋分裂模式的M。crozieri与高速多视图延时成像。利用开源斐济平台提供的图像配准和反卷积工具，可以从这些数据中重建早期开发的3D和4D（3D +时间）。我们讨论了我们的研究结果的优点和缺点的自建显微镜。我们的结论是，自制的显微镜，如OpenSPIM，连同可用的开源软件，如MicroManager和斐济，使SPIM访问任何有兴趣连续访问自己的光片显微镜。然而，构建OpenSPIM并非没有挑战，开放获取显微镜是值得的，如果重要的话，时间和金钱的投资。多视图4D显微镜比我们预期的更具挑战性。我们希望我们在这个项目中获得的经验能够帮助未来有类似抱负的OpenSPIM用户。本文的在线版本（doi：10.1186/s12861-016-0122-0）包含补充材料，可供授权用户使用。
Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory’s own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri – a non-model animal. Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions. The online version of this article (doi:10.1186/s12861-016-0122-0) contains supplementary material, which is available to authorized users.