Both darkfield microscopy and phase contrast microscopy are methods that allow the invisible to be seen without having to stain and eventually kill them. Purchasing darkfield lenses, systems, or equipment to attach to your microscope is more affordable than purchasing phase contrast equipment, but the latter generally provides better image resolution.

How Darkfield Microscopy Works
If you aren’t using a specially designed microscope that allows you to instantly view specimens with darkfield optics, you would have to attach an opaque disc below your microscope’s condenser. You can also make a pair of darkfield discs yourself by cutting out two pieces of circle made up of black electrical tape. Check if their length is equivalent to the width of your slide. If it is, stick them to the slide, making sure that there’s a considerable distance between both circles. Illumination should be evenly distributed and the aperture diaphragm opened wide.

This ensures that your naked eye won’t be engulfed with all kinds of light. The only right that would reach them is the one being scattered by the sample since darkfield optics illuminate samples from the sides rather than directly beaming light onto the sample.

It is also possible to obtain darkfield effects with a normal microscope by adjusting the position of your sample, using higher light intensity and setting your microscope at low magnification and no higher than 100x.

With darkfield microscope, all components of the sample are made visible by causing them to appear bright white against a black background. If the sample has colored components, they project colors that are different from their original shades. Using special oil-immersion lenses may also help in improve the clarity and detail of your sample.

Darkfield microscopy is especially effective when you are performing preliminary examinations of cell suspensions like that of small one cell organisms, bacteria, and cells and tissues. Human cells that are easiest to obtain are those that you can scrape from your tongue. You can also use darkfield microscopy for studying slides that are very lightly prepared and initial samples taken from soil infusions and pond water. Lastly, darkfield microscopy may also be used to determine motility or movement in tissue cultures.

There are two types of darkfield illumination: transmitted and reflected. The best way to practice your skills in darkfield microscopy is by using yeast cells as your practice sample. Explore the various darkfield microscopic techniques here until you master adjusting your microscope for darkfield experiments.

How Phase Contrast Microscopy Works
It was in 1935 that the Dutch physicist F. Zernike was able to convert phase to amplitude differences. He was later on awarded with the Nobel Prize in 1953. Among other achievements made possible by phase contrast microscopy, his work had succeeded in making mitosis not only visible but recorded in video.

Nowadays, there are more specially designed phase contrast microscopes in the market today than those with built-in optics. As mentioned earlier, phase contrast microscopy may provide better images but they are also heavier on the pocket.

You can use a normal microscope to achieve phase contrast optics by attaching certain accessories. In most cases, you would have to attach a phase contrast condenser, distinguishable from other condensers because of its ring-shaped mask, and an extra phase ring behind your microscope’s objective.

The additional phase ring is important because of two reasons. Firstly, it provides alignment or balance between the light that is and isn’t refracted. This consequently produces a dark background instead of the white one produced by normal bright field microscopes. Secondly, the phase ring ensures that the correct phase difference is achieved.

Phase contrast microscopy is nonetheless imperfect. Shade off and halo patterns are two of its greatest problems. Both are commonly referred to as image distortions and phase artifacts. Halos are caused by the use of the phase ring itself or more specifically, the small ray of diffracted light it produces. Halos may become bigger or darker due to optical path difference fluctuations.

On the other hand, shade-off occurs more often when large and extended specimens are being studied. It becomes more visible when the image intensity isn’t parallel with the specimen’s optical path difference.

While darkfield optics is better at low magnification, phase contrast microscopy works when high magnifications like 400x and 1000x are used. Most living organisms are made discernible with phase contrast microscopy. The best sample to practice phase contrast microscopy is a fresh cell suspension of Nagleria gruberi or amoeba inside a tissue culture flask.

Ultimately, using darkfield microscopy is better for preliminary research while phase contrast microscopes are ideal for more sophisticated experiments.