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 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.

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.

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

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.

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).

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.

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.

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.