荧光显微镜基础知识

Epifluorescence microscopes

You really don’t need to know in great detail about how a microscope works in order to do fluorescence imaging, but it can help a little when it comes to troubleshooting which, really, given how iterative science is, ends up being about 99.8% of the time, right? And one of the things that you probably do need to understand if you’re trying to solve an imaging problem is the filter setup for your fluorophore.

Most fluorescence imaging is done using fluorescence microscopes that have these essential components:

• A light source: usually a xenon arc or mercury vapor lamp but more recently powerful LEDs
• A filter (for incoming light): that narrows the wavelengths of the incoming light to only those used to excite the sample called, funnily enough, the excitation filter
• A dichroic beamsplitter or mirror:  to reflect the excitation light to the sample and simultaneously transmit only the emitted light from the sample back to the detector (figure below)
  
• A filter (for emitted light): that transmits only the wavelengths of the emitted light from the sample and blocks all the light passed through the excitation filter, called, you guessed it, the emission filter
• A CCD camera: Emitted light does you no good if you can’t detect it, and for fluorescence imaging the detector is usually a CCD camera. The camera is usually also connected to a computer screen, which shows you a picture of your image.

Figure 1. A dichroic beamsplitter allows longer wavelengths of light to pass through the filter while reflecting shorter wavelengths of light.

The light path in epifluorescence microscopes

The illustration below shows the typical light path of an epifluorescence microscope. Most microscopes that are used for cell biology are arranged so that the light travels through the objective lens to illuminate the sample, and then the light emitted from the sample travels back through the same objective to the detector.

Figure 2. The yellow line represents the arrangement of the light path for brightfield illumination. The illumination light does not travel through the objective, only the transmitted light from the sample. The blue line illustrates the path of excitation light, which travels through the filter cube and objective to the sample, and the resultant emission light (shown in green) simultaneously travels through the objective and filter cube and onto the detectors. In epifluorescence microscopy, both the excitation and emission light travel through the same objective.

This arrangement—where both the illuminated and emitted light travels through the same objective lens—is referred to as epifluorescence microscopy, where “epi” is borrowed from the Greek to mean “same”. A more correct term would be epifluorescence illumination, but most people assume the illumination part, since fluorescence depends on illumination. A transillumination fluorescence microscope is not as common, but you still may encounter a setup where the illumination and collection of signal are on opposite (trans) sides of the stage with the sample in between.

Figure 3. Typical light path in an epifluorescence microscope. Notice that the both excitation and emission are controlled by the dichroic, which reflects excitation light (shorter wavelengths) onto the sample and passes the resulting emission light (longer wavelengths) through the filter and on to the detector (the viewer or the camera).

Magnification vs. resolution

Magnification

It’s pretty important to understand the difference between magnification and resolution when it comes to getting a good result when you’re doing fluorescence imaging. When we talk about magnification, we are referring to how much bigger an object appears when we look at it under the microscope (Figure 4).

Resolution

In contrast, when we talk about resolution in a practical sense, we are referring to how much detail we can distinguish in our image, which can be subjective. In a more technical sense, resolution is limited by the refractive properties of light.

Limits of resolution in epifluorescence microscopy

What does that actually mean? It means that a typical epifluorescence illumination compound microscope cannot resolve or distinguish between two objects that are less than 200 nm apart. Additionally, because the whole sample is illuminated at the same time, you are detecting all of the in-focus and out-of-focus light in your sample. These limitations mean that, depending on the lenses in your objectives, you will be able to determine that two different-colored probes are present in the same cell, but you may not always be able to resolve their spatial relationship to each other without a lot of controls, individual pixel analysis, and math. Also, because you don’t have any information about depth, you can’t really draw any sound conclusions about volumes from an image taken with an epifluorescence microscope. By understanding and working within the limitations of your system, you can be confident in the data and images you collect, as well as being able to fully understand your data and formulate conclusions.

Laser scanning confocal microscopy still relies on a compound light microscope setup, but can give you more resolution. The increase in resolution comes from the use of lasers for illumination, which narrows the excitation range to ~2–3 nm. This is around 10 times narrower than the range of wavelengths you get when using excitation filters. Additionally, the ability to obtain an image from just one focal plane—while removing all of the scattered and out-of-focus light generated in an experiment—can also increase resolution. The restriction to one focal plane is accomplished using a pinhole to block out-of-focus light before it gets to the detector, referred to as optical sectioning. The pinhole permits light from only a very narrow section of the sample and gives you information about depth. This is an improvement over the resolution you can get using epifluorescence, which collects the light from many focal planes within a cell. There are other alternatives for scientists who want more resolution, but they tend to be more specialized and require greater technical knowledge to get started.

Figure 6. The resolving power of various microscopes, with representative objects within range for both light microscopes and electron microscopes.

Upright vs. inverted microscopes

You will sometimes hear people refer to microscopes as upright or inverted. These terms refer to the location of some components, like objectives and light sources. Upright microscopes have objectives placed above the stage where you put your sample; inverted microscopes have objectives below the stage where you put your sample.

There’s no fundamental difference in the ability of upright and inverted microscopes to produce and channel light along various paths. The image quality you are able to achieve will have more to do with your sample preparation, lenses, light source and wavelength, fluorophore filter set, and camera than the locations of components on the microscope. Some experiments will require a particular orientation in order to accomplish what you need, so it’s always a good idea to look at a new-to-you microscope and walk yourself physically through the steps of your experiment to make sure the setup will work for you.

Figure 7. Inverted and upright microscopes both utilize epifluorescent illumination: the main difference is the location of the objectives relative to the stage where the sample is placed.

荧光显微镜

您并不需要详细了解显微镜的工作原理才能进行荧光成像，但是这能够对故障排除有所帮助——考虑到科学研究的重复性，这大概会占到99.8%的时间。并且如果您试图解决成像问题，确实需要了解荧光基团的滤光片设置。

大部分荧光成像通过荧光显微镜完成，其包括以下基本元件：

• 光源: 通常是氙弧灯或汞灯，但近年来也使用高功率LED
• 滤光片（入射光）: 将入射光波长缩减到只留下用于激发样品的波长，有趣的是，其被称为激发滤光片
• 二向分色镜或反射镜:  将激发光反射到样品上，并同时只通过来自样品的发射光，传递到检测仪（如下图）
  
• 滤光片（发射光）: 只能通过来自样品的发射光波长，并阻挡所有通过激发滤光片的光线，如您所想，其被称为发射滤光片
• CCD照相机: 发射光如果不能检测到，则没有任何用处；对于荧光成像来说，检测仪通常是CCD照相机，这种照相机一般也连接到电脑屏幕，能够为您呈现图像。

图 1. 二向分色镜允许较长波长的光线通过滤光片，同时反射较短波长的光线。

荧光显微镜中的光路

下图展示了荧光显微镜的典型光路。大部分用于细胞生物学的显微镜都设计为让光线穿过物镜照射样品，然后来自样品的发射光再通过同一个物镜到达检测仪。

图 2. 倒置和正置显微镜都使用荧光照明：主要的区别是物镜相对于放置样品的载物台的位置。
 

照射和发射光通过同个物镜的设计被称为落射荧光显微镜（epifluorescence microscopy），词中的“epi”借用自希腊语，意为“相同”，更加准确的说法应是落射荧光照明，但是因为荧光依赖于照明，所以人们默认省略了照明部分。透射照明荧光显微镜不常见，但是您也可能遇到放置样品的载物台在中间，照射和信号收集在其两侧的设置方法。

图 3. 落射荧光显微镜的典型光路。注意激发光和发射光通过分光镜控制，其将激发光（较短波长）反射到样品上，而让得到的发射光（较长波长）通过滤光片到达检测仪（观察者或照相机）。

放大倍数与分辨率

放大倍数

为了在荧光成像中获得良好结果，理解放大倍数和分辨率的区别很重要。当提到放大倍数，指的是当我们在显微镜下观察一个对象时，它比原本放大了多少（图4）。

分辨率

与此相反，实际意义上的分辨率指的是图片中我们能够分辨多少细节，这可能是主观的。从技术层面更深入地讲，分辨率受到光的折射特性的限制。

荧光显微镜的极限分辨率

这到底是什么意思？它指典型的落射荧光照明复合显微镜无法分辨或区分距离小于200 nm的两个物体。此外，因为整个样品被同时照射，所以样品中所有聚焦和离焦的光线都被检测到。这些限制意味着，根据物镜中的镜片，您可能能够确定一个细胞中的两个不同颜色的探针，但是如果没有很多对照、单像素分析和计算，就无法分辨它们之间的空间关系。此外，因为您没有掌握任何深度相关信息，所以无法从落射荧光显微镜拍摄的图片中得到关于体积的可靠结论。通过了解系统的限制，您能够对获得的数据和图像更加确信，也能够完全理解您的数据并明确表达结论。

激光扫描共聚焦显微镜也依赖于复合光显微镜设置，但能够获得更高的分辨率。其分辨率的提高源于使用激光作为照明，其将激发光范围缩窄到~2-3nm。这比使用激发滤光片组准确十倍。此外，一次实验中只从一个焦平面获得图像，使用针孔阻挡离焦光线到达检测仪，可以去除所有分散和离焦光线，从而提高了分辨率对单个焦平面的限制，这被称为选择性分区。这种针孔只允许来自样品很窄区间的光线通过，所以能够改善深度方面的信息。这比使用落射荧光获得的分辨率有所提高，落射荧光收集一个细胞中很多焦平面的光线。科研人员也可以通过其他选择获得更高的分辨率，但是这些方法更加专业化，并需要更多的技术知识才能着手。

图 6. 不同显微镜的分辨能力，以及光学显微镜和电子显微镜视野内的代表性物体。

正置和倒置显微镜

有时候听到人们称显微镜为正置或者倒置，这些术语指的是一些元件的位置，如物镜和光源。正置显微镜的物镜在放置样品的载物台之上；倒置显微镜的物镜在放置样品的载物台之下。

正置和倒置显微镜产生和引导光线通过不同路径的能力并没有根本不同。其实您所能获得的图像质量与样品制备、镜片、光源和波长、染料滤光片组设置以及照相机等更加相关，而非显微镜元件的位置。一些实验需要特定方向才能获得所需的结果，所以观察一台没用过的显微镜并全面考虑实验步骤以确定其设置符合您的实验需要，这始终是一种明智的做法。

图 7. 黄线表示明场照明的光路设置，照射光不穿过物镜，只有来自样品的投射光穿过物镜。蓝线表示激发光线路径，它穿过滤光片和物镜到达样品，得到的发射光（显示为绿色）同时穿过物镜和滤光片并到达检测器。在落射荧光显微镜中，激发光和发射光都穿过同一个物镜。