While confocal microscopy seems to have become pervasive in cell biology, widefield microscopy techniques still have a special and important place. This month on the Microscopy and Imaging Channel, we’re focusing on widefield microscopy techniques: covering the basics of what these techniques are and when you should turn to them.
What is this ‘Widefield’ you talk of?!
What is widefield microscopy anyway? Widefield microscopy refers to techniques where the whole specimen is exposed to light. This differs from confocal microscopy where you are using a laser to illuminate only a very small part of the sample.
The age-old problem of contrast
The age-old problem in microscopy is: how do you create contrast? The majority of cellular material is relatively transparent, making it hard to distinguish features using light. There are many solutions to the contrast problem, and these many solutions have led to the many flavors of widefield microscopy. We’ll take a look at each of these in turn;
Bright field microscopy:
Remember that basic microscope that you used in high school biology? It was a simple widefield microscope, or more specifically a bright field microscope. In bright field microscopy you illuminate the sample from the bottom with white light, and observe the sample from the top (unless, of course, you are using an inverted microscope- but that is a topic for another article). The main advantages of this technique are the simplicity of the microscope system and the simplicity of sample preparation. The drawbacks? Contrast is based on the absorption of light by the cellular material. Since most cellular material absorbs light similarly, the result is a low contrast image. Using stains on your bright field sample is one way to increase contrast, although you should always keep in mind that each stain only highlights select structures!
Similar to bright field microscopy, in widefield fluorescence microscopy the microscope illuminates the sample from the bottom and observes the sample from the top. The difference is in the light. Rather than using white light, in fluorescence microscopy you use light of a specific frequency that can excite the fluorescent molecules of interest in your sample. The source of fluorescence in the sample can be naturally occurring auto-fluorescence, genetically engineered fluorescent molecules (e.g. GFP, YFP, etc), or fluorescent antibodies introduced through immunostaining. The difference in the signal from the fluorescent molecules and the sample’s background auto-fluorescence provides the contrast for the image. For a really great introduction to fluorophores, read this article from the Bitesize Bio archives.
Phase contrast microscopy:
Samples that are relatively transparent by bright field microscopy can often be imaged by phase contrast microscopy. In bright field microscopy the contrast is derived from the difference in light absorption by the different cellular materials. However, the sample doesn’t only absorb light- it also refracts light! Differences in the refractive index across the sample result in different light phase shifts. Phase contrast microscopy translates these phase shifts into contrast in the final image. While phase contrast microscopy provides great contrast, but doesn’t work well for thick samples and can produce halo artifacts around structures. Phase contrast microscopy is covered in greater depth in this Bitesize Bio article.
Dark field microscopy:
In dark field microscopy, greater contrast is created in the image by collecting only the light that is scattered by the sample. Because the background does not scatter much light compared to the sample, dark field images have a characteristically dark (almost black) background while the samples themselves appear bright (or vice versa). Your first impression may be that a dark field image is just the negative of a bright field image; take heed, different structures may be visible with each technique! Curious about how you collect only the light that is scattered by the sample? Check out the Bitesize Bio dark field microscopy article later this month.
Differential interference contrast (DIC):
If a lab has the set-up, DIC is generally the method of choice for imaging unstained low-contrast samples. This technique uses polarized light which the microscopy splits into two orthogonal beams and then sends through the sample. When the two orthogonal beams are recombined, phase shifts which occurred in each individual beam as it traveled through the sample lead to interference, and this interference is translated into contrast in the final image. DIC provides an excellent image with good resolution, but is not suitable for thick samples or highly pigmented cells. There is so much more to learn about this fascinating technique: be sure to read the DIC article coming up later this month!
That’s it for today’s tasting of widefield microscopy. Although we have covered all the major widefield techniques, there are still more flavors out there to explore. I hope it has whetted your appetite!