Phase Contrast

This is the setup for phase contrast the Olympus BX and CH microscopes.

After the invention of the achromat lens the second most significant development in microscopy was phase contrast. This system allows the microscopist to "cheat" the classic optical tradeoff of resolution for contrast. So you are able to have resolution and contrast at the same time. In addition, it also allows for the examination of live specimens. Before phase contrast specimens had to be stained, with some dye that produces contrast by absorbing light. Since, the dye attaches itself to a particular biological molecule, it generally renders it lifeless. Most of these stains used in biology are toxic to the specimen they stain. The reason phase contrast is not toxic to the specimen is that it does not generate contrast by absorbing light, which is the way most contrast is formed. So there are no stains required. Phase contrast uses interference to generate contrast. Light has wave properties in addition to particle properties. Each waveform can be described as having an amplitude and frequency. The amplitude of a light wave is the size of the peaks on the waveform. If the amplitude increases the light appears brighter. The frequency deals with the number of wavelength that passes a fixed point in a unit of time. Since, light moves at the same speed it is dependent on the wavelength. For example, a wavelength of light at 550 nanometers corresponds to green light. Interference contrast works by first separating a beam of light into two components. Each of the component waveforms would have only half the amplitude of the original wave. If one of the beams of light is passed through a material of a different refractive index then the light will be slightly retarded. When the two waveforms are brought back together the waves no longer match. The peak of the first wave matches with the trough of the second wave. The two waves then cancel each other out, producing a waveform with very low amplitude. This is called destructive interference, and will make the image of the specimen appear dark. Likewise, if the peaks are aligned on both the waveforms, there will be constructive interference and the image will be bright. The amplitude of the "in phase" waveforms will be sum of the two.

Most phase contrast microscopes used today actually use two ways to produce non-absorption contrast. Interference contrast and stop contrast. In stop contrast an annulus slot is placed below the condenser lens. The cone of light that passes through the specimen is refracted slightly more in material that has a higher index of refraction. In the objective there is another annulus. If the specimen did not refract the light, it would be able to make it through the object annulus and ultimate to your eye. However if it was refracted it would not pass and the specimen would appear dark. In a typical phase contrast microscope a phase plate replaces the objective annulus. This phase plate is specifically designed to setback the wave front to maximize the contrast for biological specimens. Needless to say the optics must be perfectly aligned for all this to work. The correct slide and cover glass must be used and the specimen cannot exceed the design index of refraction. The microscope that we use in this lab has the phase plates designed to be used with particulate cellular specimens in an aqueous environment.

1. Select the phase annuli turret in the condenser lens assembly to "O" for brightfield operation.

2. Using a specimen with the appropriate slide and cover glass, set up the microscope for Kohler illumination. This must be done correctly or phase contrast will not work.

If you don't remember how to setup Kohlor illumination , click here.

3. Select the either the 10X, 20X, 40X or 100X objective.

4. Depending on which objective you are using, select the appropriate phase annulus from the table below.

Objective	BX40 Phase Turret	BH Phase Turret
4X	N/A	N/A
10X	Ph1	10
20X	Ph1	20
40X	Ph2	40
100X oil	Ph3	100

5. Open the iris diaphragm all the way.

6. Remove the eyepiece, and visually check the annulus and the phase plate are aligned. If not, use the annulus alignment knobs located on the condenser lens assembly to correct the alignment.

7. Replace the eyepiece.