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Unstained and therefore transparent preparations that do not absorb light are called «phase samples», as they slightly alter the light that passes through them. Usually, the phase-contrast delays 1/4 wavelength with respect to direct light and without deviation, which passes through a stained sample. Unfortunately, the eyes, like the sensors of the cameras, are not able to appreciate these phase differences. The human eye is only sensitive to the colors of the visible spectrum (variations in the frequency of light) or of different levels of light intensity (variations in the amplitude of the light wave).
In phase objects, the «zero point» of the light passes through or around the sample without any deviation. The light diffracted by the object is not reduced in amplitude (as in opaque samples), but is «delayed», slow (phase delay) due to the function of the refractive index and the thickness of the sample. This diffracted light, decreased around 1/4 wavelength, reaches the image plane with the light deflected, but in interference, essentially decreased in intensity. The result is that the image at eye level lacks contrast to make visible the almost invisible details.
Zernike was successful in developing a method, now known as phase-contrast microscopy, which allows to obtain clear images of transparent objects as if they were opaque samples.
A schematic illustration of the configuration of a phase-contrast microscope is shown in Figure 1.
The example shows excellent contrast when the diffracted light and direct light (not deflected) are delayed 1/2 wavelength. The Zernike phase method consists of accelerating the direct light 1/4 wavelength, so the wave difference between direct light and the diffused light of a transparent sample is precisely 1/2 wavelength. As a result, the two components of light (direct and diffracted) produce «destructive» interference in the eyepiece. This method produces an image with darker details on a lighter background. This type is known as positive phase-contrast (Figure 2).
A second option is to delay the direct light 1/4 wavelength, in this way both lights (direct and diffracted) produce «constructive» interference in the eyepiece. This method produces an image with lighter sample details on a dark background. This type is known as negative phase-contrast. (Figure 3)
The accessories required for phase-contrast operation are a condenser placed under the stage, equipped with phase rings, and a set of phase-contrast lenses, each with internal phase ring. It also includes a green filter (to increase the resolution) and a phase telescopic eyepiece (to center the rings).
Phase microscopy techniques are particularly useful with thin samples distributed in the visual field. There are certain limitations in this technique:
- Phase images are usually affected by haloes around the cells. These haloes are optical «artifacts», which can sometimes obscure the boundaries between adjacent samples.
- Phase rings limit the numerical aperture of the optical system, thereby reducing the resolution.
- Phase-contrast does not work with thick samples, because phase variations occur in areas just above or below the focus plane.
- Phase-contrast images can appear gray if white or green light is used if a green filter is used.