Microscope Resolution

Not to be confused with magnification, microscope resolution is the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities.

If the two points are closer together than your resolution then they will appear ill-defined and their positions will be inexact. A microscope may offer high magnification, but if the lenses are of poor quality the resulting poor resolution will degrade the image quality.

Below is Abbe's equation in order to calculate approximate resolving power:

resolving power = wavelength of light used/2 (numerical aperture of objective)

Numerical Aperture

microscope resolution showing lenses

Microscope resolution is affected by several elements. An optical microscope set on a high magnification may produce an image that is blurred and yet it is still at the maximum resolution of the objective lens.

The numerical aperture of the objective lens affects the resolution. This number indicates the ability of the lens to gather light and resolve a point at a fixed distance from the lens.

The smallest point that can be resolved by an objective is in proportion to the wavelength of the light being gathered, divided by the numerical aperture number. Consequently, a higher number corresponds to a greater ability of a lens to define a distinct point in the view field.

The numerical aperture of an objective lens also depends on the amount of optical aberration correction.

Aberration and Diffraction

Aberration is another factor related to lens performance that impacts resolution. Simply stated, a microscope’s lenses are designed to focus light rays on a single point.

More light rays straying from this focal point will occasion a greater amount of aberration and a greater amount of diffraction. However, if all light rays are focused on one infinite pinpoint this will also cause diffraction.

Diffraction in microscopy is nothing more than interference or noise caused by the light rays passing through and around the specimen being viewed, passing through the small aperture of a lens, or bending at the edges of the objective.

Without diffraction the specimen would not be visible. However, too much diffraction limits the resolution of a microscope.

Lens manufacturers work to design lenses with the highest aberration correction possible for a particular class of objective lens.

Mathematical computations have proven that the smallest point of focus for light rays without causing diffraction is 200 nanometers. This is the ideal resolution for an optical microscope.

Also, to increase resolution the condenser’s aperture number must be matched to the objective and it must be properly adjusted so that the light rays transmitted through it form a precise light cone illuminating the specimen.

If the light cone is not properly formed diffraction will increase.

Light Wavelength and Refractive Index

Microscope resolution is also impacted by the wavelength of light being used to illuminate the specimen. Longer wavelengths of light offer less resolution than short wavelength illumination.

Near-ultraviolet light has the shortest usable wavelength and offers the greatest resolution. Following near-ultraviolet in descending order of wavelength are red, orange, yellow, green, blue and violet.

The range in nanometers of the wavelength of the visible light is from 380nm to 750nm.

Another method of improving microscope resolution is to increase the refractive index between the objective lens and the specimen.

The refractive index is merely a ratio expression of the relative speed of light passing through a substance as a proportion of the speed of light in a vacuum.

As the refractive index increases the speed of the light passing through a medium is slower. As light slows down the wavelength gets shorter and yields better resolution.

Objective lenses are manufactured that allow imaging in immersion oil which has a refractive index of 1.51 and substantially shortens light waves.

Microscope resolution is the most important determinant of how well a microscope will perform and is determined by the numerical aperture and light wavelength.

It is not impacted by magnification but does determine the useful magnification of a microscope.

Even though 200 nanometers is considered the optimal resolution for optical microscopes, higher resolutions can be obtained using fluorescence microscopy.

When combined with a laser light source, focal plane resolution of 15-20 nanometers can be achieved. However, in routine microscopic observations the highest resolution possible is usually unnecessary. 

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