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Microscope Resolution 101: The Numerical Aperture and Light Wavelength

A microscope is a wonderful and invaluable tool that enables us to see things far beyond what the naked eye can see. Now, everything can be magnified to an indefinite amount of time. In fact, we have even invented microscopes that can let us see at the subatomic level.

There’s just one problem. Enlarging an image over and over more often than not results into a blurry and distorted image. Therefore, we can say that magnification alone is not directly proportional to image resolution.

But wait, what is resolution, and how do we fix this problem?

What is microscope resolution?

Microscope resolutionWhile we all know that magnification is essentially how big an image can appear to be, the meaning of the term resolution is not as clear to many.

Resolution, also called resolving power, is largely defined as the amount of detail clearly visible in an image. More specifically, microscope resolution refers to the minimum distance between any two points of a specimen that can be distinguished as separate entities.

Simply put, it’s how clear or blurry an image is.

What factors affect the microscope’s resolution?

This resolving power intrinsically depends on the properties of the light source being used to illuminate the specimen- namely, the wavelength of the light and the size of the opening of the microscope’s optical components.

Of course, there are a few other factors that come into play. These are dependent on the composition of the microscope and the quality of the specimen being used. We will go over these in a bit.

First, let’s talk about the numerical aperture and light wavelength.

Numerical aperture

The resolution of a microscope mainly depends on the numerical aperture (NA) of the condenser and the objective lens. These are two of the microscope’s optical components where light passes through to illuminate the specimen.

This numerical aperture is the range of angles in which an optical component can accept or emit light. To put it more simply, it’s how big of a cone of light can pass through the lenses of the microscope. The higher this number, the better the resolution of the magnified specimen image.

Think of it this way- when you look through a tiny hole like, say, a drinking straw, you can’t see as much detail as when you look through a bigger pipe-sized hole.

So, we can say that a high numerical aperture results in a high-resolution image.

Light wavelength

The wavelength of light is defined as the distance between any two successive crests or troughs of the light wave. A shorter wavelength is typically directly proportional to a higher frequency, meaning, more light travels in a shorter amount of time.

Light wavelength is normally measured in nanometers (nm). The visible light spectrum (think rainbow) has a wavelength range of 400 to 700 nm, where violet light has the shortest wavelength and red light has the longest.

When it comes to microscopy, what you want is as much light to reach the eyepiece as fast as possible, so you can see more, and more importantly, see better. Ergo, the resolution of a microscope can be improved by changing the wavelength of light.

A shorter light wavelength produces a higher resolution image.

The Concept of Numerical Aperture for Obejectives and Condensers
Source

Other factors

Now, as we have mentioned earlier, aside from the numerical aperture of the condenser and the objective lens, as well as the light wavelength of the light from the illuminator, there are other factors that affect how resolved the microscope can be. 

For one thing, all of the optical components of the microscope must be perfectly aligned and working together harmoniously. Even better, the microscope should have some capability when it comes to correcting aberrations and image distortions.

Even the specimen somewhat determines how well its magnified image can be resolved. For the best results, the specimen should be in good quality, its contrast and pigmentation should be high, and its density should be low.

Therefore…

Taking everything into consideration, we can say that:

High numerical aperture (NA) + short wavelength (nm) = high resolution

This is true regardless of whether the magnification is high or low, but what’s important to remember is the more you increase the magnification level, you should also increase the numerical aperture and use a shorter light wavelength if you want to maintain the resolution.

Some of the many ways this can be achieved is by using better quality lenses, such as oil immersion objective lenses with a high refractive index, as well as using light sources with shorter wavelengths, such as ultraviolet light, X-rays, and gamma rays.

What microscope should you use?

As you have probably inferred from everything we have discussed so far, there’s only so much you can do to control the numerical aperture and the light source of a microscope. While there are adjustment knobs that give you some leeway, those only work up to a certain point.

This is why it’s important that you use the correct type of microscope for your specimen to begin with. This is because different types of microscopes have different capabilities in terms of magnification and image resolution.

Compound microscopes

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Most, if not all, compound light microscopes utilize a conventional light source such as halogen, incandescent, and LED bulbs, and even mirrors. All these illuminators only have the capacity to produce visible light. As such, compound microscopes only have a resolution of around 1.2 nanometers, similar to a confocal microscope. 

The problem is that when a specimen (or parts of a specimen) has a density of more than 200 nanometers, visible light is not enough to produce an image with enough detail and clarity. Even if you use a high end compound light microscope with oil immersion lenses, there’s only so much it can do once you reach its maximum magnification level.

Electron microscopes

As a point of comparison, dissecting microscopes only have a resolution of 120 nanometers, whereas electron microscopes range from 10 to 0.2 nanometers, depending on whether you use a scanning electron microscope (SEM) or a transmission electron microscope (TEM).

These electron microscopes do not use conventional light to magnify the specimen, rather, a beam of supercharged electrons is what does the job. Electron microscopes are what can give you the most detailed specimen images possible.

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