All You Need To Know About Microscope Resolution

Microscope resolution is how much information can be determined from light refraction through a specimen plane which is then displayed to the viewer using a microscope.

You can also think of resolution as the minimum distance between two visible points the observer can pick out.

For instance, if you were examining plant matter through a microscope, a pair of cells next to each other would look blurry until the lens is focused.

Once you focus the microscope, the plant cells would look clearer, so you’d be able to tell the difference between each cell. 

It can be hard to understand what microscope resolution is as it involves many different elements, including wave optics, aperture, and airy disk patterns. 

We’ll look at some of these principles in more detail below, so that you can understand more about what resolution means within microscopy terms.

What Are Wave Optics?

If you want to understand more about resolution, you need to understand what wave optics are. The suggestion that light travels as light waves was proposed by the Dutch creator, Christiaan Huygens.

He was born in 1629 and lived until 1695. Huygens was also an astronomer who was well versed in math and physics.

He presumed that waves come in two main kinds, spherical and plane. 

It can be difficult to picture what plane waves look like, but try to think of light moving in a straight line. As the light travels directly forward, think of waves moving in sequence afterward. 

Spherical waves, as the name implies, have a curved form. These waves will either approach a spot or deviate from one, looking like the curve of a sphere as they do so. 

Like plane waves, it can be hard to picture what spherical waves look like, but don’t worry if you don’t understand just yet. 

Huygens theorized that both plane and spherical waves are made from fundamental components. These are known as wavelets, which are small spots of light that send out light in all directions. 

A plane wave may have wavelets that travel directly forwards, while the wavelets from spherical waves move inwards and travel out instead.

It’s important to understand what wavelets are as they are significant within the topic of diffraction. 

If there is a particularly tiny opening, and a plane wave is behind it, attempting to travel out, only a certain number of wavelets may pass through.

The ones that can move through the hole will transmit light outwards in several directions, known as diffraction. Diffraction can tell us why light looks like it bends and moves in every direction. 

Now that we know what wavelets and diffraction are, we can see how microscopes use these phenomena. Microscopes send light through a sample to be examined.

As it does so, before the light can get to the objective lens, the light is in a diverging spherical waveform. 

These spherical waves will be transformed into plane waves by the objective lens.

These waves will turn back into spherical ones by the microscope’s tube lens, but instead of diverging, they will now converge towards a point. 

Objectives with a greater numerical aperture can capture more of these waves than objectives with a lower numerical aperture.

We’ll talk about what numerical aperture means and its role in resolution in the following section. 

What Does Numerical Aperture Mean?

Numerical aperture sounds complicated, but the term only means how well an objective lens can collect light data.

This involves information about the sample we’re viewing and any resolution details at a specific working distance. 

As mentioned in the previous section, microscopes work by sending light up from a light source through a condenser lens.

The light will then move through the lens’ hole, through a slide, then through smaller holes, and around the sample. 

This causes diffraction, resulting in an upside-down cone of light. How well the objective lens can collect or capture this light is what we mean when discussing numerical aperture. 

We can calculate numerical aperture through formulas and calculations using the light cone. The formula for this is: 

NA (Numerical Aperture) = n(sin µ)

Now we can look at this formula in more detail. The term µ will be half of A, the angular aperture. This means that we can calculate µ by dividing the angle’s cone by 2.

The term n refers to the refractive index of the medium that lies between the sample’s slide cover and the objective lens. 

A medium is an object that light will have to move through. For example, air is a baseline, which has a refractive index of 1.00.

If you use a medium that’s thicker, like immersion oil, the refractive index will be higher.

Applying immersion oil will mean that the objective lens touches the oil that lies on the slide cover, resulting in less light refracting out. 

Wavelets dispersing on higher numerical apertures will show more information as there’s room between the wavelets.

The reverse is through for wavelets dispersing on lower numerical apertures, as less detail will be visible. 

What Are Airy Disks?

We know that microscopes send light through a sample which then turns into a visible picture. The spots on the sample are visible as smaller images known as Airy patterns.

The center maximum of these patterns is known as an Airy disk, which resembles concentric darker and lighter circles. 

The English mathematician and astronomer, George Biddell Airy, was born in 1801 and lived until 1892.

He was the first person to come up with the Airy Disk notion, and expanded on his ideas in his paper, ‘On the Diffraction of an Object-Glass with Circular Aperture.’ 

Now we can go back to our first mention of resolution meaning the smallest possible distance between two specific points. These two spots are the Airy disks. 

We can determine these clear points as we travel from left to right. Resolution can then be measured at the point where we can see two elements as individual items. 

Resolution also involves understanding what the Rayleigh Criterion means.

This is more advanced mathematics, but for the sake of reason, it’s simply a mathematical measure for when two spots can be identified differently from each other. 

Now that we’ve covered numerical aperture, the Rayleigh criterion, and Airy disks, we can theorize that smaller Airy disks result in better resolution, resulting in more detail. 

Airy disks and numerical aperture are related in an inverse relationship. As the numerical aperture increases, more light passes through, which leads to smaller Airy disks. 

Understanding Resolution As A Whole

You may have come across the numerical aperture in terms of magnification, which may make you wonder how numerical aperture, refractive index, and light waves are so important in resolution. 

Resolution is known as the shortest possible distance between two points. We measure this distance in micrometers. The formula for resolution is as follows:

r = λ/(2NA)

The term r means resolution, λ refers to wavelength, while NA means numerical aperture.

As mentioned above, the objective lens and the condenser should have the same numerical aperture, which means we can multiply this by 2. 

As we already know, the formula for numerical aperture is:

 NA = (n) sin(µ)

The term n is the refractive index of the medium which light is passing through. The term µ is half of the light cone’s angular aperture.

If you aren’t using oil to examine something, the refractive index will be around 1, which is the refractive index of air. We can the work out resolution with the following formula:

R = λ /2((n) sin(µ))

We can alter the wavelength’s value by using color filters, which will change the numerator’s value within the equation.

Using oil immersion and switching the objective lens can also change the refractive index and numerical aperture values within the denominator. 

Another point to note is that as the numerical aperture is important within magnification, you may wonder how magnification and resolution are connected.

We can work out the resolution from the above equation, which will give us a measurement in micrometers.

If you can work out the resolution, you’ll be able to identify the shortest distance between two points of the sample. 

However, if this distance isn’t large enough, our eyes won’t be able to notice any space between these points, resulting in a blurry picture. 

No matter what magnification level you use, there will be a maximum resolution you’ll be able to reach, due to the elements needed to calculate resolution. 

For a visual observation to occur, the minimum magnification required is around 500 multiplied by the objective lens numerical aperture.

The maximum magnification will be 1000 multiplied by the same numerical aperture. 

How To Increase Resolution?

Most modern microscopes deliver effective resolution, as long as the right samples are being viewed. Despite this, various ways can help to increase resolving power. 

Clean The Objective Lens And Specimen Plane

Just a fingerprint on the air objective’s front lens is enough to affect a distinct picture reproduction of a sample.

Smudges and marks can affect the way light scatters out from the lens. 

This also applies when using immersion lenses that still have emulsion or resin residue left over, like water or oil. If this occurs, use a soft cloth and lens cleaner to carefully clean away any residue. 

If you don’t have lens cleaner available, pure ethanol will do, just remember that it is flammable if you are working in a lab setting. 

Check The Thickness Of The Coverslips

Coverslip thickness is thought of when creating objective lenses. These coverslips should have the same high aperture of the objectives.

If a different coverslip thickness is used, the optical image’s quality will deteriorate. 

In most cases, objectives that have a numerical aperture of over 0.7 can manage a 10-micrometer variation.

Objectives with a lower numerical aperture, around 0.3 – 0.7, can handle higher variation levels, reaching up to 30 micrometers. 

Use The Right Immersion Oil

Every objective that has a numerical aperture larger than 0.95 can be used with immersion substances.

These are usually oils with a refractive index of 1.515. If any air bubbles reach the immersion substance, the image’s quality will go down. 

You need to apply the oil carefully to prevent bubbles from forming. You can identify any bubbles by taking off the eyepiece, then looking at the objective lens’ rear focal plane. You can also use a Bertrand lens to do this.

If you do notice any air bubbles, you’ll need to clean the sample plane and the objective. You should then re-apply the oil, taking care when doing so. 

Conclusion

A microscope’s resolving power is how well it can determine very small details within a sample to be examined. 

The term resolution is used often in everyday language, which makes it seem like a simple topic.

However, as noted above, microscope resolution involves various elements that affect how well a microscope depicts an image.

Resolution involves wave optics, numerical aperture, and Airy disks. 

The numerical aperture is also involved in magnification, which is also connected to resolution.

However, no matter what magnification level you use, the maximum resolution will have a limit depending on the refractive index, light wavelength, and numerical aperture that are used. 

There are things you can do to increase resolving power, like checking that the objective is free from residue, and using the right immersion oil.

You should also ensure that your cover slips aren’t too thick or thin. 

Frequently Asked Questions

What Factors Affect The Resolution Of A Microscope?

The main factor that affects a microscope’s resolution is the numerical aperture of an objective. The resolution also depends on the type of sample being viewed, light intensity, and variance correction.

Other elements, like contrast-enhancing technology, can be used within the sample itself, or the microscope’s optical components. 

Does Resolution Increase With Magnification?

Resolution improvement relies on numerical aperture increases, not a magnification increase. Optical resolution completely relies on objective lenses, which is different from digital resolution within cameras.

In this case, digital resolution relies on a camera’s objective lens, camera sensor, and monitor. 

How Does Wavelength Affect Resolution?

Light wavelengths are significant elements when determining a microscope’s resolution. Shorter wavelengths deliver higher resolution compared to longer ones.

The best resolution power used in optical microscopes uses almost ultraviolet light, which has shorter wavelengths compared to visible light. 

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