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Phase Contrast Microscopy

12/4/16


Phase Contrast Microscopy

Phase contrast objective lenses and a matching condenser are needed to accomplish phase contrast microscopy. Phase contrast is valuable because unstained objects have little effect on the amplitude of light waves, and there is no color in the object, making bright field observing ineffective. However, light passing through a relatively thick or dense part of a cell, such as the nucleus, is retarded compared with light passing through media only or a thin part of the specimen. The degree of retardation and the extent of the phase shift that results are proportional to the thickness of the object. The phase contrast microscope exploits the interference effects that take place when the two types of light recombine. If the waves complement each other the light is brighter, while if they are 180 degrees out of phase they cancel each other and the image is dark.

To use phase contrast, first align the microscope in bright field, using a prepared slide. Replace the prepared slide with the slide intended for phase contrast viewing.

Focus on the specimen if you can find it. If not, focus on an air bubble or piece of debris that is clearly in the same focal plane as the specimen. Place the microscope in phase contrast mode, by rotating the condenser turret or using the appropriate insert. Phase rings must match, so check the objective and condenser mode. A phase 3 lens, for example, must be used with a matching phase 3 ring.

Use the phase centering screws to obtain the best contrast. An effective way is to rotate one screw until the image has the best possible contrast, usually when the background is darkest. Then rotate the other to improve the effect. Alternatively, you can line up the phase rings by replacing an eyepiece with a phase telescope. Focus the telescope (not the microscope) on the rings and adjust the centering screws to make them concentric.

Phase contrast shows remarkable detail, such as features of nuclei within specimens as well as at their edges. It is very useful for observing extremely thin objects such as cilia and flagella, or bacteria.


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