How A Phase Contrast Microscope Works
Phase contrast is an optical technique that allows users to view microorganisms more clearly. If you want to know how a phase contrast microscope works, you need to understand the principles behind phase contrast microscopy first.
What Happens in Phase Contrast Microscopy A phase contrast microscope allows users to view microorganisms clearly even in unstained slides. Staining them will not however prevent users from seeing the microbes clearly; if anything, it will only make the microorganisms more visible.
When light passes through an object, contact with the object will cause its phase and amplitude to change. The resulting changes will greatly depend on the object’s characteristics. Amplitude changes can lead to light absorption and consequently the emergence of colors, depending on the wavelengths involved. As for phase changes, these carry an enormous amount of vital data, but not all of it can be processed by the naked eye.
Ordinary light microscopes suffer from the same limitations. To make the data produced by phase changes present, phase contrast microscopy combine the light traveling through the object with another reference. The first person to have tried this experiment out was Frits Zernike, who later on won the Nobel Prize for his achievements in phase contrast microscopy.
Specimen Preparation Tips When Using a Phase Contrast Microscope Although you need not stain a slide for viewing specimens with a phase contrast microscope, there are still preparation steps you have to take to view the specimen with the greatest possible clarity.
Growing Cells – One way of viewing living cells with a phase contrast microscope is by “growing” them. With this technique, you place the cells in a cell culture container. Place a cover slip on it. The cell culture will ensure that your specimen will have everything it needs to grow healthily, divide, and reproduce. When your cells have reached optimal population size, remove the cover slip and turn it upside down facing a glass slide. In most cases, you’ll be using an inverted microscope with phase contrast lenses.
Fixing Cells – Certain specimens need to be “fixed” to retain the original morphology or structure of cells and tissues. If you’re using the right fixatives and you apply them properly, you could fix cells in a state almost akin to living. You can fix cells by drying or fixing them as well as using appropriate chemicals.
Sectioning Specimen – Thin sections are taken samples that are too thick to allow light to pass through its form. In the past, sections were manually cut off from the specimen with the use of a plain razor blade but these didn’t result into ideally shaped sections. Thankfully, however, modern technology has produced ways to produce thin sections easily and accurately. A microtome, for instance, can cut thin sections from a sample automatically. If you’re using a frozen specimen, you’ll need to use a cryostat to keep the specimen frozen while cutting out a thin section.
Setting Up an Ordinary Microscope for Phase Contrast Microscopy Make sure that you have all the pertinent parts ready: a phase contrast kit is generally made up of phase contrast objective lenses, a sub-condenser, and a centering telescope.
Start by adjusting the set screws to align the phase ring properly. Adjust the power of your objective lenses. Look for the switch to turn on the Bright Field (BF) mode for your microscope. Consider this as the starting point of observing specimen. BF is also referred to as the “no phase” setting.
With the microscope set at BF, start adjusting your microscope’s eyepiece to focus properly on your specimen. Use the condenser knob to adjust the condenser’s height. Change the setting of your microscope’s condenser turret to phase contrast. You can now remove the specimen to move on to the next level of adjustment.
Adjust your microscope’s condenser to center setting. Remove one of the eyepieces of your microscope and replace it with the centering telescope from your phase contrast kit. Check the resulting view. You should be seeing two circles by now. If they’re not concentric, try adjusting it until they share the same center.
If the two rings are already concentric, you can remove the centering telescope and return the original eyepiece in place. Return the specimen in place as well and fix it in place with your microscope’s stage clips.
Note: If you are using a dark field microscope, the resulting image will feature your specimen set against a dark background.
Now that you know how a phase contrast microscope works, you’ll be capable of solving all possible problems regarding the microscope’s use.
