Confocal Microscopy: Principles and Modern Practices.
Confocal Microscopy: Principles and Modern Practices.
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DOI：
10.1002/cpcy.68
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发表时间：
2020-03-01
影响因子：
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通讯作者：
Elliott, Amicia D
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文献类型：
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作者：
Elliott, Amicia D
在光学显微镜中，照明光在视场上尽可能均匀地穿过样品。对于较厚的样本，物镜没有足够的焦深，也将检测到来自焦平面上方和下方样本平面的光。失焦的光线会使图像变得模糊，从而降低分辨率。在荧光显微镜下，视野中的任何染料分子都会受到刺激，包括那些在焦外平面上的染料分子。共焦显微镜提供了一种排除来自探测器的失焦光的方法，这样它就不会对正在采集的图像造成模糊。这项技术允许在厚组织中进行高分辨率成像。在共焦显微镜中，照明和检测光学聚焦在样品中相同的衍射限制光点上，这是探测器在共焦扫描过程中唯一成像的光点。要生成完整的图像，必须将斑点移动到样品上，并逐点收集数据。共焦显微镜的一个显著优点是提供了光学切片，这允许从高分辨率的图像堆叠中三维重建样本。为此已经开发了几种类型的共焦显微镜，每种都有不同的优点和缺点。本文简要介绍了共焦显微镜。©2019 John Wiley&Sons，Inc.
In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.