Biomedical Engineering Reference
In-Depth Information
refractive index variation that is visualized, very large phase shifts can produce contrast
inversion, and halo and shade-off artifacts produce phase shift independent contrast. The
halo effect also limits the accuracy of edge definition.
Thick objects with large optical path length differences to the surround wave may produce
reduced contrast because the total phase shift of a particular region may be far from the
λ
/4
produced by diffraction and around which the phase plate is designed. Very thick samples
may also present aberrations from out-of-focus regions and so contrast is reduced. Again
the nonquantitative nature of phase contrast is apparent.
The effective numerical aperture of phase optics tends to be lower than other contrast
methods (but this does give good depth of field which is convenient for uses such as
inspection of tissue culture cells). This limits the sectioning ability of phase contrast which
is frequently seen to be less good than in DIC. The fixed nature of the condenser ring
annulus also means the condenser aperture is smaller than in brightfield or DIC and the
condenser cannot be adjusted to balance contrast and resolution.
The phase plate in the objective does have a small effect on light such as fluorescence
when the lens is used for other purposes than transmitted light phase contrast formation.
The overall attenuation of the light is quite small, but the ring tends to be relatively close to
the edge of the objective in a region of the Fourier transform of the image that contains the
higher spatial frequencies which are normally present at low amplitudes.
The fixed nature of a particular phase objective does not provide great variability for
samples that behave very differently than a typical range of biological samples the objective
is designed for.
Other transmitted light contrast methods present different advantages and disadvantages.
Understanding and using these methods offers a wealth of possibilities for visualizing
interesting biological objects. The ideas presented in the other chapters of this topic present
great promise for expanding these tools.
References
[1] D.B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging, Wiley-Liss, New York, 2001.
[2] F. Zernike, How I discovered phase contrast, Science 121 (1955) 345 349.
[3] S. Inou ´ , K.R. Spring, Video Microscopy: The Fundamentals, 2nd Ed., Springer-Verlag, New York, 1997.
[4] Phase contrast microscopy, in: E.M. Slayter (Ed.), Optical Methods in Biology, Wiley-Interscience,
New York, 1970.
Further Reading
MicroscopyU— , http://www.microscopyu.com/articles/phasecontrast/index.html . —a detailed coverage of
phase contrast and other microscopy. The Interactive Java tutorials are great educational tools.
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