Confocal Microscopy: Principles and Modern Practices

Q1 Health Professions Current Protocols in Cytometry Pub Date : 2019-12-26 DOI:10.1002/cpcy.68
Amicia D. Elliott
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引用次数: 93

Abstract

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.

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共聚焦显微镜:原理和现代实践
在光学显微镜中,照明光在视场上尽可能均匀地通过样品。对于较厚的样品,物镜没有足够的聚焦深度,来自焦平面上下的样品平面的光也会被检测到。失焦光会使图像模糊,降低分辨率。在荧光显微镜中,视场中的任何染料分子都会受到刺激,包括那些在失焦平面中的染料分子。共聚焦显微镜提供了一种方法来拒绝来自探测器的失焦光,这样它就不会对所收集的图像造成模糊。这项技术允许在厚组织中进行高分辨率成像。在共聚焦显微镜中,照明和检测光学聚焦在样品中相同的衍射极限点上,这是探测器在共聚焦扫描过程中成像的唯一点。为了生成完整的图像,必须将斑点移动到样本和逐点收集的数据上。共聚焦显微镜的一个显著优点是提供了光学切片,它允许从高分辨率图像堆栈中对样品进行3D重建。几种类型的共聚焦显微镜为此目的而开发,每种都有不同的优点和缺点。这篇文章提供了一个简明的介绍共聚焦显微镜。©2019 by John Wiley &儿子,Inc。
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来源期刊
Current Protocols in Cytometry
Current Protocols in Cytometry Health Professions-Medical Laboratory Technology
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期刊介绍: Published in affiliation with the International Society for Advancement of Cytometry, Current Protocols in Cytometry is a "best practices" collection that distills and organizes the absolute latest techniques from the top cytometry labs and specialists worldwide. It is the most complete set of peer-reviewed protocols for flow and image cytometry available.
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