Rottenfusser Rudi
Carl Zeiss Microscopy, LLC, Woods Hole, Falmouth, Massachusetts, USA.
Methods Cell Biol. 2013;114:43-67. doi: 10.1016/B978-0-12-407761-4.00003-8.
The light microscope is merely the first element of an imaging system in a research facility. Such a system may include high-speed and/or high-resolution image acquisition capabilities, confocal technologies, and super-resolution methods of various types. Yet more than ever, the proverb "garbage in-garbage out" remains a fact. Image manipulations may be used to conceal a suboptimal microscope setup, but an artifact-free image can only be obtained when the microscope is optimally aligned, both mechanically and optically. Something else is often overlooked in the quest to get the best image out of the microscope: Proper sample preparation! The microscope optics can only do its job when its design criteria are matched to the specimen or vice versa. The specimen itself, the mounting medium, the cover slip, and the type of immersion medium (if applicable) are all part of the total optical makeup. To get the best results out of a microscope, understanding the functions of all of its variable components is important. Only then one knows how to optimize these components for the intended application. Different approaches might be chosen to discuss all of the microscope's components. We decided to follow the light path which starts with the light source and ends at the camera or the eyepieces. To add more transparency to this sequence, the section up to the microscope stage was called the "Illuminating Section", to be followed by the "Imaging Section" which starts with the microscope objective. After understanding the various components, we can start "working with the microscope." To get the best resolution and contrast from the microscope, the practice of "Koehler Illumination" should be understood and followed by every serious microscopist. Step-by-step instructions as well as illustrations of the beam path in an upright and inverted microscope are included in this chapter. A few practical considerations are listed in Section 3.
光学显微镜仅仅是研究机构中成像系统的第一个元件。这样的系统可能包括高速和/或高分辨率图像采集能力、共聚焦技术以及各种类型的超分辨率方法。然而，“输入垃圾，输出垃圾”这句谚语比以往任何时候都更适用。图像操作可能被用来掩盖次优的显微镜设置，但只有当显微镜在机械和光学上都处于最佳对准时，才能获得无伪像的图像。在追求从显微镜获得最佳图像的过程中，还有一些东西常常被忽视：正确的样品制备！只有当显微镜的设计标准与标本相匹配时，或者反之亦然，显微镜光学元件才能发挥其作用。标本本身、 mounting介质、盖玻片以及浸没介质的类型（如适用）都是整个光学构成的一部分。为了从显微镜获得最佳结果，了解其所有可变组件的功能很重要。只有这样，人们才知道如何针对预期应用优化这些组件。可以选择不同的方法来讨论显微镜的所有组件。我们决定沿着从光源开始并在相机或目镜处结束的光路进行讨论。为了使这个顺序更清晰，直到显微镜载物台的部分被称为“照明部分”，随后是从显微镜物镜开始的“成像部分”。在了解了各种组件之后，我们就可以开始“使用显微镜”了。为了从显微镜获得最佳分辨率和对比度，每个认真的显微镜工作者都应该理解并遵循“柯勒照明”的操作方法。本章包括了在正立和倒置显微镜中的光束路径的分步说明以及图示。第3节列出了一些实际考虑因素。
