Dark Field versus Phase Contrast
Both dark field and phase contrast microscopy are used to illuminate samples or specimens that normally don’t show up under normal bright light. This could be either due to their transparent characteristics and absence of natural pigmentation or because they’re not just thick enough to absorb light. Dark field optics are mainly preferred over the other because they’re more affordable while some choose phase contrast illumination because they’re more powerful.
What is Phase Contrast Microscopy?
Fritz Zerknike, a Dutch physicist, is credited as the one who invented phase contrast microscopy. This illumination technique is capable of making extremely thin or transparent samples and specimens visible by use of contrast levels.
With a typical microscope, light travels through a sample or specimen. Its passage through it makes its phase and amplitude alter. The degree of change relies largely on what kind of specimen is being observed. Amplitude changes also cause colors to appear differently once the light waves pass through the objectives, reaching the eyepieces and finally the naked eye. When the sample is transparent like bacteria or too thin to absorb light such as thin sections of a particular tissue, the phase variations are difficult to discern. That’s when phase contrast microscopy comes in. A phase contrast microscope combines the light traveling through the specimen with a reference, making the changes in phase visible to the human eye.
Some microscopes have built-in capabilities for phase contrast illumination. If your microscope doesn’t have one, you can purchase a phase contrast kit and attach them to your microscope. A phase contrast kit is basically made up of two things: a phase plate and a specially designed annular diaphragm.
What is Dark Field Microscopy?
Dark field microscopy was invented to enable people to study samples and specimens that are invisible or with insufficient thickness for bright field illumination to show. A dark field microscope makes invisible appear brightly against a dark backdrop by filtering the light beams that pass through the objectives and eyepieces to reach the naked eye. Many microscopes today are capable of dark field illumination but if yours isn’t so, it would cost you little to purchase the opaque disk necessary for attaining dark field illumination. See http://www.darkfield-microscope.com for more information.
Dark Field versus Phase Contrast Microscopy
Ease of Use – Dark field microscopy is definitely easier to set up and integrate if you are using an ordinary microscope. All you have to do is just insert or attach a disk underneath the lens of your microscope’s condenser. It’s as easy as that! With phase contrast microscopy, however, there are several adjustments to make with the condenser itself and another accessory piece to attach before you can start observing invisible samples and specimen.
Costs – Enhancing your microscope with dark field optics is less costly than acquiring a phase contrast kit. Dark field optics is also easier and cheaper to maintain. If you seldom study living cells and other invisible specimen, investing in a phase contrast kit is unnecessary.
Flaws – Both dark field and phase contrast illumination techniques are not perfect. They have their own sets of advantages and disadvantages and you’ll have to consider and apply them to your situation in order to find the one most suitable for you.
A dark field microscope has very low contrast levels. In most cases, specimens must be killed before they can be placed under the microscope. Staining techniques are often used to make specimens more visible but again, this could cause their death. Also, low contrast levels require the use of extremely strong illumination. High intensity levels for light can damage specimens, too. Some specimens also have to be fixated or preserved first and again, this means killing the specimen.
Lastly, the US Department of Food and Drug Administration does not approve of live blood cell analysis with the use of dark field illumination so you’re facing another limitation if you are practicing in the field of medicinal science.
On the other hand, a phase contrast microscope is capable of studying invisible specimens without damaging them. In that area, they are better than dark field microscopes. They do not fare similarly well, however, when it comes to the quality of their images. Dark field optics rarely produces images with artifacts while it’s the opposite for phase contrast microscopy.
In phase contrast microscopy, there are two common image problems and they’re halos and shade-off effects. Certain light distributions cause halos to appear in images. These are occasionally helpful to the user because they can highlight contrast differences or make the specimen more visible, especially the parts forming the specimen’s borders or edges, but they’re mostly undesirable. There are ways of reducing halos, but it’s tedious to have to do so in the first place. Shade-off appears most often with large specimens.
All in all, consider your needs and the common types of specimens you’re studying to determine whether you need phase contrast or dark field optics for your microscope.
