Basic building units and properties of a fluorescence single plane illumination microscope
Basic building units and properties of a fluorescence single plane illumination microscope
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DOI：
10.1063/1.2428277
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发表时间：
2007-02-01
影响因子：
1.6
通讯作者：
Stelzer, E. H. K.
中科院分区：
文献类型：
--
作者：
Greger, K.;Swoger, J.;Stelzer, E. H. K.
所有荧光显微镜的关键问题是荧光团的有效使用，即从激发的荧光团中检测尽可能多的光子，以及仅激发聚焦的荧光团。这个问题在 EMBL 实施的基于光片的显微镜 [单平面照明显微镜 (SPIM)] 中得到解决，该显微镜仅照亮检测物镜焦平面中的荧光团。光片是一个在一个方向上被准直并在另一个方向上聚焦的光束。由于在检测器焦平面之外没有荧光团被激发，因此该方法还提供了固有的光学切片。与共焦荧光显微镜相比，可观察时间点的总数可以提高几个数量级。实际的改进系数取决于获取的平面数量以及实现特定信噪比所需的平面数量。 SPIM 由五个基本单元组成，分别解决 (1) 光检测、(2) 样本照明、(3) 生成适当的光束、(4) 样本的平移和旋转，以及最后 (5) 不同机械和电子部件的控制、数据收集和数据后处理。我们首先描述 EMBL SPIM 的基本构建单元及其最相关的属性。然后，我们介绍了该仪器的基本原理及其独特的性能，例如荧光团的有效利用、减少的光毒性效应、真正的光学切片能力和出色的轴向分辨率。我们还讨论了如何实现各向同性分辨率。详细解释了光学设置、控制硬件和控制方案。我们还描述了基本设置的一些不太明显的改进，这些改进可以提高性能。该仪器的特性通过使用 EMBL 的 SPIM 之一成像的生物样本图像得到证明。(c) 2007 年美国物理研究所。
The critical issue of all fluorescence microscopes is the efficient use of the fluorophores, i.e., to detect as many photons from the excited fluorophores as possible, as well as to excite only the fluorophores that are in focus. This issue is addressed in EMBL's implementation of a light sheet based microscope [single plane illumination microscope (SPIM)], which illuminates only the fluorophores in the focal plane of the detection objective lens. The light sheet is a beam that is collimated in one and focused in the other direction. Since no fluorophores are excited outside the detectors' focal plane, the method also provides intrinsic optical sectioning. The total number of observable time points can be improved by several orders of magnitude when compared to a confocal fluorescence microscope. The actual improvement factor depends on the number of planes acquired and required to achieve a certain signal to noise ratio. A SPIM consists of five basic units, which address (1) light detection, (2) illumination of the specimen, (3) generation of an appropriate beam of light, (4) translation and rotation of the specimen, and finally (5) control of different mechanical and electronic parts, data collection, and postprocessing of the data. We first describe the basic building units of EMBL's SPIM and its most relevant properties. We then cover the basic principles underlying this instrument and its unique properties such as the efficient usage of the fluorophores, the reduced photo toxic effects, the true optical sectioning capability, and the excellent axial resolution. We also discuss how an isotropic resolution can be achieved. The optical setup, the control hardware, and the control scheme are explained in detail. We also describe some less obvious refinements of the basic setup that result in an improved performance. The properties of the instrument are demonstrated by images of biological samples that were imaged with one of EMBL's SPIMs.(c) 2007 American Institute of Physics.