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Microscopes

Microscopes are used in laboratories to magnify samples, such as cells and tissues, so we can see structures that would not be possible to observe with the naked eye. There are many different types of microscopes but the main types are light microscopes, the transmission electron microscope (TEM), and the scanning electron microscope (SEM).There are many other microscopes used in…

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# Microscopes

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Microscopes are used in laboratories to magnify samples, such as cells and tissues, so we can see structures that would not be possible to observe with the naked eye. There are many different types of microscopes but the main types are light microscopes, the transmission electron microscope (TEM), and the scanning electron microscope (SEM).

There are many other microscopes used in laboratories; light and electron microscopes are only two examples! Other types include X-ray microscopes, scanning probe microscopes, and scanning acoustic microscopes.

## Microscope Magnification and Resolution

There are two factors that are extremely important when looking at a structure using a microscope, and these factors are:

• Magnification
• Resolution

Magnification refers to how much an object has been enlarged.

Resolution describes the ability of a microscope to distinguish two close points (objects) from each other, i.e. see detail.

Magnification can be calculated by using the following equation:

Magnification = $\frac{lengthofimage}{actuallength}$

You can also rearrange the equation accordingly to find out what you are looking for.

Suppose we want to calculate the actual length of a cheek cell. We are using the magnification at 12,500X and the length of the cheek cell under the microscope is 10 mm.

Let's first convert 10 mm into µm which is 10,000 µm (remember 1 mm = 1,000 µm).

Let's now rearrange our equation to calculate the actual length. This gives us the length of the image/magnification. When we insert our values into the rearrange equation, it gives us:

Actual length = 10,000/12,500 = 0.8 µm

Light microscopes have a lower ability to magnify objects without affecting the resolution. Light microscope magnification can reach 1,000-1,500X. If we compare these values to electron microscopes, the magnification can reach 1,000,000X!

For resolution, light microscopes can reach only 200nm, while electron microscopes can achieve an impressive 0.2 nm. What a difference!

## Light microscope diagram

Light microscopes magnify objects by using two biconcave lenses that manipulate the light falling into the lenses, making them appear bigger. The light is manipulated by a series of glass lenses that will focus the beam of light onto or through a specific object.

Fig. 1 - The different parts of a light microscope

## Parts of a light microscope

Although light microscopes may have slightly different parts according to different models and manufacturers, they will all contain the following general features.

### The stage

This is the platform where you will place your specimen (usually on a glass slide). You can position the specimen in place by using the stage holder clips.

A specimen refers to a living (or previously alive) organism or a part of a living organism used for scientific study and display.

### Objective lens

The objective lenses will gather the light reflected from your specimen to magnify the image.

### Eyepiece (with ocular lenses)

This is the point at which you observe your image. The eyepiece contains ocular lenses, and this magnifies the image that is produced by the objective lens.

### Coarse and fine adjustment knobs

You can adjust the focus of your magnified image using coarse and fine adjustment knobs on the microscope.

### The light source

The light source, also often referred to as the illuminator, provides artificial light to illuminate your specimen. You can use the light intensity control to adjust the strength of the light beam.

## Types of electron microscopes (EM)

Unlike light microscopes, electron microscopes use electron beams to magnify the image of specimens. There are two main types of EMs:

• Transmission electron microscope (TEM)
• Scanning electron microscope (SEM)

### Transmission electron microscope (TEM)

TEM is used to generate cross-sectional images of specimens at high resolution (up to 0.17 nm) and with high magnification (up to x 2,000,000).

Fig. 2 - Parts of the electron transmission microscope

Take a look at Fig. 2 to familiarize yourself with the different parts of TEM.

Electrons carrying a high voltage are fired via electron gun at the top of TEM and travel through a vacuum tube. Instead of using a simple glass lens, TEM uses an electromagnetic lens which is able to focus electrons into an extremely fine beam. The beam will either scatter or hit the fluorescent screen located at the bottom of the microscope. Different parts of the specimen will show up on the screen depending on their density and pictures can be taken using the camera fitted near the fluorescent screen.

The specimen studied needs to be extremely thin when using TEM. In order to do so, samples undergo a special preparation before being cut with an ultramicrotome, which is a device that uses a diamond knife to generate ultra-thin sections.

The size of a mitochondrion is between 0.5-3 um, which could be seen in a light microscope. In order to see inside a mitochondrion, you need an electron microscope.

### Scanning electron microscope (SEM)

SEM and TEM are similar in some ways as they both use an electron source and electromagnetic lenses. However, the main difference is how they create their final images. SEM will detect reflected or ‘knocked-off’ electrons, while TEM uses electrons transmitted to show an image.

SEM is often used to show the 3D structure of the surface of a specimen, while TEM will be used to show the inside (such as the inside of a mitochondrion mentioned earlier).

Flower pollen is around 10–70 µm (depending on species) in diameter. You may think that you could see it under the naked eye but what you will see are random clusters. Individual pollen grains are much too small to be seen under a naked eye! Although you might be able to see individual grains under a light microscope, you will not be able to see the structure of the surface.

When using SEM, pollen can appear in different shapes and have a varied rough surface. Have a look at Fig. 3.

Fig. 3 - Pollen of common flowering plants.

## Specimen preparation for microscopy

Your sample specimen must be prepared carefully in order for your microscope of choice to correctly produce a magnified image.

### Preparation for light microscopy

In light microscopy, the two main ways to prepare your sample are wet mounts and fixed specimens. To prepare a wet mount, the specimen is simply placed on a glass slide, and a drop of water is added (often a cover slide is placed on top to fix it in place). For fixed specimens, your sample is attached to the slide using heat or chemicals and the cover slide is placed on top. To use heat, the specimen is placed on the slide which is gently heated over a heat source, like a Bunsen burner. To chemically fix your sample, you can add reagents such as ethanol and formaldehyde.

Fig. 4 - A Bunsen burner

### Preparation for electron microscopy

In electron microscopy, specimen preparation is more difficult. Initially, the specimen needs to be chemically fixed and dehydrated to become stable. This needs to be done as soon as possible when removed from its environment (where an organism has lived or if a cell, from the body of an organism) to prevent changes to its structure (e.g. changes in lipids and deprivation of oxygen). Instead of fixing, samples can also be frozen, then the specimen is able to retain water.

Aside from this, SEM and TEM will have different steps of preparation after the initial fixing/freezing. For TEM, the specimens are suspended in resin, which makes it easier to slice and cut into thin cross-sections using an ultramicrotome. Samples are also treated with heavy metals to increase the contrast of the image. The regions of your specimen that have readily taken up these heavy metals will appear darker in the final image.

As SEM produces an image of the surface of a specimen, the samples are not cut but rather coated with heavy metals, such as gold or gold-palladium. Without this coat, samples can start to build up too many electrons which leads to artefacts in your final image.

Artefacts describe structures in your specimen that do not represent the normal morphology. These artefacts are produced during specimen preparation.

## Field of view of microscopes

The field of view (FOV) in a microscope describes the observable area in your ocular lenses. Let's have a look at some example FOVs with different specimens (Fig. 5 and 6).

Fig. 5 - An aplacophoran.

Fig. 6 - An ostracod.

Let's learn more about who is in Fig. 5 and 6! These particular organisms come from benthic deep-water Angola samples which were obtained using a grab (Fig. 7).

Fig. 5 shows an aplacophoran which, at first glance, looks like a hairy worm. However, it is in fact, a mollusc, meaning they are related to squids and octopuses! Aplocophorans are not well known since they live in the deep. Most can reach about 5cm (some species, even 30cm) in length.

Fig. 6 shows an ostracod (seed shrimp), which looks like a bivalve but is actually a crustacean. This means that they are related to crabs and lobsters. They are extremely small in size and usually don't get bigger than 1mm. Their shrimp-like flesh is protected by two shells, hence the initial look of a bivalve.

Fig. 7 - A grab being deployed to obtain deep water samples

There is a simple formula that you can use to find out the FOV:

$FOV=\frac{Fieldnumber}{Magnification}$

The field number is usually on the ocular lens next to the ocular magnification.

If your field number is 20 mm and your magnification is x 400 you can calculate the FOV by inputting your values into the equation:

FOV = 20 / 400 = 0.05 mm!

## Microscopes - Key takeaways

• Magnification and resolution determine how the image will be seen through the ocular lenses. They are inter-linked.
• The light microscope is the main microscope used to teach students.
• The transmission electron microscope and the scanning electron microscope are often used by scientists to investigate very small structures.
• Electron microscopes have a much higher resolution compared to light microscopes.
• Field of view of the microscope is the image that you can see when looking through the ocular lens(es).

## References

2. Fig. 5 - Epimenia verrucosa (Nierstrasz, 1902) at Osaka Museum of Natural History. Accepted name is Epimenia babai Salvini-Plawen, 1997 (https://upload.wikimedia.org/wikipedia/commons/d/d9/Epimenia_verrucosa.jpg) by Show_ryu is licensed by CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0/deed.en)

Magnification = length of image/actual length

Microscopes work by using multiple concave lenses that make images appear bigger.

Light microscopes use two types of lenses: objective and ocular.

Objective lenses gather reflected light from your specimen to magnify the image. Ocular lenses simply magnify the image produced by the objective lens.

There are many types of microscopes but five examples include:

1. Light microscope
2. Electron microscopes
3. X-ray microscope
4. Scanning probe microscope
5. Scanning acoustic microscope

Transmission electron microscope (TEM) and scanning electron microscope (SEM).

## Microscopes Quiz - Teste dein Wissen

Question

Identify three types of microscopes used in laboratories.

Light microscope

Scanning electron microscope

Transmission electron microscope

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Question

Which microscope is most commonly used?

The light microscope

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Question

Which types of microscope have a higher resolution?

Electron microscopes

Show question

Question

Define magnification and resolution.

Magnification refers to the degree that an object has been enlarged. Resolution describes the ability of a microscope to distinguish two close points (objects) from each other.

Show question

Question

Light microscopes have a higher resolution than electron microscopes. True or False?

False.

Show question

Question

What is the equation used to calculate magnification?

Magnification = Length of an image / Actual length of an image

Show question

Question

What equation would you use to calculate an actual length of an image when you know the magnification and the length of an image (in the field of view)?

Length of an image / Magnification

Show question

Question

What are the names of two lenses in the light microscope?

Occular and objective lenses.

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Question

What are the two ways you will be able to manipulate (i.e. move around) the specimen when on the stage?

By hand or using stage controls.

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Question

What is the name of the slide placed on top of the specimen?

Cover slide (or slip).

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Question

How can you adjust resolution on the light microscope?

Using the coarse and fine adjustment knobs.

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Question

To see the image, light is used in the light microscopy and an _______ _______ is used in the electron microscopy.

Electron beam.

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Question

How are samples stained in preparation for electron microscopy?

Using heavy metals, such as gold or gold-palladium.

Show question

Question

What is the equation to calculate field of view?

Field of view = Field number / Magnification

Show question

Question

Identify two samples of chemicals used to chemically fix your sample in preparation for light microscopy.

Ethanol and formaldehyde.

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