Different microscope observation techniques explained

Countless occupations –­ from forensic science to engineering – use microscopes. However, they don’t all use microscopes in the same way.

The difference lies in technique. Modern microscopes are able to use a range of special techniques and equipment to get a better look at hard-to-see samples and specimens. 

Let’s take a quick look into how some of the more common microscopy techniques work.

Brightfield

Using a brightfield microscope is the simplest and most common technique – you may even have used it in school.

In this case, light is shone through a sample and into the magnifying lenses. The light is blocked by the denser parts of the sample, which shows up as darker object against a white background.

It’s a very easy technique to set up and use but it has problems with low contrast, particularly with transparent and colourless specimens.   

Darkfield

Using a darkfield microscope for darkfield microscopy is similar to brightfield microscopy, but it provides better contrast for transparent and colourless specimens.

Like brightfield, light is shone through a sample, but darkfield microscopy uses indirect light instead of direct light. A dark disk is placed in the path of the light so that only obliquely angled light gets around it.

Because of the angle, the light ordinarily wouldn’t enter the lens and you would only see the black background of the disk.

But when the angled light hits a specimen, it gets refracted or reflected in different directions and can enter the microscope lens where it can be seen. This results in brightly lit specimens against the dark background of the disk.

Phase contrast

Phase contrast microscopy is a more advanced technique used for enhancing contrast, particularly with transparent samples.

Phase contrast microscopes involves the use of special objective lenses and a specific kind of condenser. This approach is again similar to brightfield where light is shone through a sample, but this setup takes it a step further. 

As light passes through the specimens, it is slightly refracted. The microscope converts this tiny difference in light wavelength into visible contrast. This makes it much easier to see transparent specimens and their internal structures.

A big advantage of phase contrast is that it doesn’t harm living specimens in any way, so it’s great for observing microorganisms and cells in their natural habitat.

DIC

Differential interference contrast (DIC) microscopy is another advanced technique for enhancing contrast.

In a DIC setup, polarized light is shone into a special prism that splits the light into two separate beams with different polarizations. Both beams are then passed through the specimen, where their optical pathways are altered by the refractive indices and thickness of the specimen.

The two beams then enter a second prism followed by a second polarizer, which recombines them back into a single beam.

When they are merged, the differences between the two beams interfere with each other, altering the wavelength of the light. These wavelength changes are then converted into visible colour and contrast.

Fluorescence

Fluorescence microscopy uses fluorescent substances to provide an extremely sensitive and reliable imaging technique.

The principle is fairly simple – shine a light onto a fluorescent substance and it will re-emit that light at a longer wavelength. Fluorescence microscopy takes advantage of this with the use of fluorescent dyes.

The target cell, molecule or protein is stained with a fluorescent dye, then hit with a very bright light. The light is then filtered so that only the fluorescent wavelengths are allowed through, creating a colourful and very specific image.

Using a fluorescence microscope is particularly useful for observing cells and molecules and is often used to help visualise dynamic cellular processes.

Polarized light

Polarized light microscopy refers to a few different techniques that involve the use of polarized light.

As well as being used in some specialised techniques such as DIC, polarized light is useful for observing reflective and birefringent materials.

In quality assurance and electronics repair, microscopes are often equipped with a pair of polarizing filters that can be adjusted to control the amount and polarity of light that gets through. This can be very useful when looking at anything metallic or reflective as the glare from the lighting can hide important details.

In geological fields and materials science, polarized light microscopes are used to identify and analyse substances with an optical quality known as birefringence.

Birefringent materials have two different refractive indices when exposed to light of different polarities. By using a pair of polarizing filters, you can adjust the polarity of the light passing through the sample and get a better look at its structure and optical qualities.

Hopefully, you now have a working understanding of the main techniques that make microscopes such an endlessly fantastic tool.