In microscopy, the numerical aperture of an optical system such as an objective lens is defined by:
where n is the index of refraction of the medium:
- n = 1.00 for air
- n = 1.33 for pure water
- n = 1.52 for immersion oil
and θ is the half-angle of the maximum cone of light that can enter or exit the lens.
What is microscope resolution and how is it connected to the numerical aperture?
Simply put, when light hits an object, it diffracts.
A single beam of light will be split into several different diffraction orders. The same type of thing happens when light hits a microscopic specimen; the diffraction orders spread out.
The bigger the cone of light brought into the lens, the more of these diffraction orders which can be collected by it. So, it has more information to form a resultant image and the higher the resolving power of the lens will be.
The bigger a cone of light that can be brought into the lens, the higher its numerical aperture is.
Therefore the higher the numerical aperture of a lens, the better the resolution of a specimen will be which can be obtained with that lens.
In microscopy, NA is important because it indicates the resolving power of a lens.
The size of the finest detail that can be resolved is proportional to
where λ is the wavelength of the light.
A lens with a larger numerical aperture will be able to visualize finer details than a lens with a smaller numerical aperture.
Increasing the magnification and the numerical aperture of the objective reduces the working distance, i.e. the distance between front lens and specimen.
How to obtain higher numerical apertures?
In practice, it is difficult to achieve numerical aperture values above 0.95 with dry objectives.
Higher numerical apertures can be obtained by increasing the imaging medium refractive index (n) between the specimen and the objective front lens.
Microscope objectives are now available that allow imaging in alternative media such as:
- Water (refractive index = 1.33),
- Glycerin (refractive index = 1.47), and
- Immersion oil (refractive index = 1.52).
A majority of objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oil.
In practice, however, most oil immersion objectives have a maximum numerical aperture of 1.4, with the most common numerical apertures ranging from 1.0 to 1.35.
Immersion Oil contributes to two characteristics of the image viewed through the microscope:
- finer resolution and
These characteristics are most critical under high magnification; so it is only the higher power, short focus, objectives that are usually designed for oil immersion.
Oil immersion objectives are generally available from 40 to 120x. These must not be confused with “high dry” objectives or water immersion objectives that are also made in this range.
Just as an “oil” immersion objective must be used with oil to get a usable image, a “water” immersion objective must be used with water and a “dry” must be used dry.
The use of oil on a high dry will destroy the image by negating corrections for spherical and chromatic aberration.