Optika Italy B-380 Manuel D'utilisation page 3

Introduction to phase contrast
Unstained specimens that do not absorb light are called phase objects because they slightly alter the phase of
the light diffracted by the specimen, usually by retarding such light approximately 1/4 wavelength as compared
to the undeviated direct light passing through or around the specimen unaffected. Unfortunately, our eyes as well
as camera film, are unable to detect these phase differences. To reiterate, the human eye is sensitive only to the
colors of the visible spectrum (variations in light frequency) or to differing levels
of light intensity (variations in wave amplitude).
In phase specimens, the direct zeroth order light passes through or around the specimen undeviated. However,
the light diffracted by the specimen is not reduced in amplitude as it is in a light-absorbing object, but is slowed
by the specimen because of the specimen's refractive index or thickness (or both). This diffracted light, lagging
behind by approximately 1/4 wavelength, arrives at the image plane out of step (also termed out of phase) with
the undeviated light but, in interference, essentially undiminished in intensity. The result is that the image at the
eyepiece level is so lacking in contrast as to make the details almost invisible.
Zernike succeeded in devising a method–now known as Phase Contrast microscopy–for making unstained,
phase objects yield contrast images as if they were amplitude objects.
A schematic illustration of the basic phase contrast microscope configuration is illustrated in Figure 1.
Phase
Ring
Deflected
light
Objective
Specimen
Condenser
Annular
Ring
Light from
source
Figure 1
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