Understanding Numerical Aperture in Microscopy

The importance of numerical aperture in microscopy.

Transcript: In this video, we will discuss the importance of numerical aperture in microscopy.
There are several basic principles in microscopy that are important to consider when using a light microscope to observe specimens or small objects. Such principles include magnification, resolution, and numerical aperture. NA is a critical factor to consider as it typically changes with the magnification of your lens, and it can significantly affect resolution and illumination.
By definition, numerical aperture is a unitless value that describes the ability of an objective lens to collect light to resolve specimens viewed through a microscope at a specific distance. Lenses with higher NA can gather more light, producing brighter and more resolved images.
Numerical Aperture, often written as NA, can be calculated by multiplying the refractive index, n, of the medium between the lens and coverslip by the sin of theta. And theta is the angular aperture or half of the angle of the cone of light that a lens can collect.
You will notice in the equation that the value of the NA is proportional to the refractive index. Therefore, one way to increase NA, specifically when working with higher magnification lenses, is to use a medium with a greater refractive index than air, such as immersion oil.
Immersion oil reduces light refraction so that more angles of light from the specimen reach the objective lens. What this means is that when light must travel up from the coverslip into a medium with a different refractive index (like air), the light path will bends at the border between these two mediums, which can result in less light reaching the objective lens and ultimately lowering the resolution and image quality.
Immersion oil has a similar refractive index to the coverslip, so refraction is minimized.
Objective lenses typically have the NA value printed on their barrel, as shown here for a ten and 100x objective lens. It is also important to note that lenses that require an immersion oil will also have that printed on the barrel. Be aware that there are multiple types of immersion oils, so be sure you use the most suitable for your applications.

References:
1. Davidson, Michael. “Numerical Aperture.” Nikon's MicroscopyU, https://www.microscopyu.com/microscop....

2. Abramowitz , Mortimer, and Michael Davidson. “Numerical Aperture and Resolution.” Anatomy of a Microscope - Numerical Aperture and Resolution | Olympus LS, https://www.olympus-lifescience.com/e....

3. Waters, Jennifer. “Numerical Aperture.” YouTube, YouTube, 28 Nov. 2018,    • Numerical Aperture  .

Video made by Emily Bosdachin.