Light and resolution in epifluorescence microscopes
By placing your sample on the stage and directing light through it, the filters, detectors, and camera, you will be able to visualize and capture an image of your sample.
Learn about basic light path and filter configurations, what governs the limit of resolution, and the differences between upright and inverted scopes.
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. 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.
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 epifluorecence 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
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).
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.
Figure 4. Two 6 μm beads taken at 3 different magnifications, 4x, 10x, and 40x.
Figure 5. Same images matched in size to show differences in resolution.
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. 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.