Phase contrast microscope

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A phase contrast microscope is a microscope that does not require staining to view the slide. This microscope made it possible to study the cell cycle.

Phase condenser

As light travels through a medium other than vacuum, interaction with this medium causes its amplitude and phase to change in a way which depends on properties of the medium. Changes in amplitude give rise to familiar absorption of light which gives rise to colours when it is wavelength dependent. The human eye measures only the energy of light arriving on the retina, so changes in phase are not easily observed, yet often these changes in phase carry a large amount of information.

The same holds in a typical microscope, i.e., although the phase variations introduced by the sample are preserved by the instrument (at least in the limit of the perfect imaging instrument) this information is lost in the process which measures the light. In order to make phase variations observable, it is necessary to combine the light passing through the sample with a reference so that the resulting interference reveals the phase structure of the sample.

This was first realized by Frits Zernike during his study of diffraction gratings. During these studies he appreciated both that it is necessary to interfere with a reference beam, and that to maximise the contrast achieved with the technique, it is necessary to introduce a phase shift to this reference so that the no-phase-change condition gives rise to completely destructive interference.

He later realised that the same technique can be applied to optical microscopy. The necessary phase shift is introduced by rings etched accurately onto glass plates so that they introduce the required phase shift when inserted into the optical path of the microscope. When in use, this technique allows phase of the light passing through the object under study to be inferred from the intensity of the image produced by the microscope. This is the phase-contrast technique.

In optical microscopy many objects such as cell parts in protozoans, bacteria and sperm tails are essentially fully transparent unless stained (and therefore killed). The difference in densities and composition within these objects however often give rise to changes in the phase of light passing through them, hence they are sometimes called "phase objects". Using the phase-contrast technique makes these structures visible and allows their study with the specimen still alive.

This phase contrast technique proved to be such an advancement in microscopy that Zernike was awarded the Nobel prize (physics) in 1953.

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[edit] References

Bennett, A., Osterberg, H, Jupnik, H. and Richards, O., Phase Microscopy: Principles and Applications, John Wiley and Sons, Inc., New York, 320 pages (1951).
Zernike, F., Phase-contrast, a new method for microscopic observation of transparent objects. Part I.., Physica: 9, 686-698 (1942).
Zernike, F., Phase-contrast, a new method for microscopic observation of transparent objects. Part II.., Physica: 9, 974-986 (1942).
Zernike, F., How I discovered phase contrast., Science: 121, 345-349 (1955).
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