Rubbing Your Microscope’s Eyes: A Guide to Optical Resolution

Ever wake up especially groggy in the morning, finding it takes a few minutes and a few eye rubs to be able to decipher the numbers on your alarm clock? Our eyes have the ability to resolve an image, so that you can observe separate objects and details. Similarly, microscopes have a parameter of resolution: how good it is at being able to distinguish between separate objects. More technically speaking, resolution has to do with the minimum distance at which two (or more) points can be viewed as individual points.

Crazy formula warning!

As with all things related to microscopy, there is a crazy formula to define this distance. If you really want to know, it is called the Rayleigh Criterion and looks like this:

But, as we pointed out in the Introduction– you don’t need a degree in physics to understand microscopy. So, what does that formula mean for the average biological scientist who wants to create pretty images?

Thankfully, this formula shows that resolution really only depends on two things;

(1)The lambda in the formula is the wavelength of light which is emitted by the imaging instrument to illuminate the sample.

(2)NA is the numerical aperture of the microscope objective. In other words, the ability of the objective to capture light over a range of angles. A small r value depends on a large NA and a short wavelength. A small r value also means that two points can be closer together and still be distinguished as two points- high resolution!

Not so airy-faerie..

Resolution also has to do with the diffraction pattern of the light surrounding these different points, which is given the amusing name of Airy patterns (with all due respect to Sir Airy). These Airy patterns look like a bull’s eye, a ripple pattern from a stone thrown into a calm lake, or the Target store logo (for our US readers!). The centre point of illumination in this pattern is the Airy disk, and the disk together with the “ripples” makes up the Airy pattern.

The two determinants of optical resolution are…

Above, when the term “point” was used, it should technically be subbed with “Airy disk.” Because Airy patterns are larger than the actual object point, they are harder to resolve—the patterns radiating from two disks can overlap even if the points are physically separate. Other than this technicality of nomenclature, the implications of the formula are the same: light and NA are the two determinants of optical resolution.

How to rub the eyes of your microscope

In conclusion, the factors that you can control in your microscope system (that is, how you can rub your microscope’s eyes) to affect resolution include:

• Wavelength of emitted light—affects r value
• Aperture diameter—affects NA and size of Airy disks
• Condenser and objective NA—affects r value
• Specimen contrast
• Uniform specimen illumination

Please let us know if you would like us to expand on any of the topics mentioned above- it’s a lot to take in we know!