(1) Phase reversal When n'n, the contrast between the light and dark of the image is reversed, which is called phase reversal. When the phase difference δ = 0, it is unrecognizable. As the δ increases, the contrast becomes larger. When δ continues to increase to a certain value, phase reversal occurs. With a 90% high absorbance (high contrast) objective, this transition is about 0.55 λ, which is about 0.33 λ with an objective with 70% standard absorbance. Objectives with higher absorbance values ​​should be used to resolve smaller optical path differences.
(2) Halo and fade effect In the imaging process of phase contrast microscopy, when a structure becomes dark due to the phase delay, it is not the loss of light, but the result of redistribution of light on the image plane. Therefore, light that clearly disappears in the dark area will have a bright halo around the darker object. This is a shortcoming of the phase contrast microscope, which hinders the observation of the fine structure, and the halo phenomenon is more serious when the annular diaphragm is narrow. Another phenomenon of phase contrast microscopy is the dimming effect, which means that when the phase difference is observed in a large area with the same phase delay, a contrast drop occurs at the edge of the region.
(3) Influence of sample thickness When the phase difference observation is performed, the thickness of the sample should be 5 μm or less. When a thicker sample is used, the upper layer of the sample is clear, and the deep layer is blurred and phase shift occurs. Interference and scattering interference of light.
(4) The influence of coverslips and slides must be covered with a cover slip, otherwise the bright ring of the annular diaphragm and the dark ring of the phase plate are difficult to overlap. Phase contrast observation also has high requirements on the glass quality of slides and coverslips. When there are scratches, uneven thickness or unevenness, bright ring skew and phase interference will occur. In addition, if the slide is too thick or too thin, the ring-shaped bright ring will become larger or smaller.
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