Several Notes and Tips for Using Phase Contrast Microscopy
Use of Phase Contrast Microscopy
1) Install the phase contrast device: remove the condenser lens of the ordinary microscope, and install the condenser lens with an annular diaphragm on a matching microscope; remove the ordinary objective lens of the microscope, and replace it with an objective lens with a phase plate; replace the eyepiece with Focusing eyepieces for phase contrast microscopes; special green filters (sometimes not necessary) are placed at the light entrance of the light source.
2) Light alignment and focus: Adjust the focusing eyepiece and the thickness adjustment screw until a white circle and a black circle can be clearly seen in the field of view. The black circle is fixed, and the white circle can be moved by adjusting the screw on the condenser device, so that the black circle and the white circle completely coincide. Take off the focusing eyepiece and replace it with the original eyepiece of the microscope to check.
Analysis of problems in the use of phase contrast microscope
1. The influence of sample thickness When performing phase contrast observation, the thickness of the sample should be 5 μm or thinner. When using a thicker sample, the upper layer of the sample is very clear, but the deep layer will be blurred and cause phase shift interference. and light scattering.
2. The influence of cover glass and slide glass The sample must be covered with a cover glass, 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 slide glass and cover glass. When there are scratches, uneven thickness or unevenness, bright ring distortion and phase interference will occur. In addition, when the slide is too thick or too thin, the bright ring of the annular diaphragm will become larger or smaller.
3. Halo and fading effect In the imaging process of phase contrast microscope, when a certain structure becomes dark due to phase delay, it is not the loss of light, but the result of redistribution of light on the image plane. So light that is apparently lost in dark areas will appear as a bright halo around darker objects. This is a disadvantage of phase contrast microscopy, which hinders the observation of fine structures, and the halo phenomenon is more serious when the annular diaphragm is very narrow. Another phenomenon in phase-contrast microscopy is the shading effect, which refers to the loss of contrast at the edges of a larger area with the same phase delay when observed by phase contrast.
4. Phase inversion When n'n, the light and dark contrast of the image is just opposite, which is called phase inversion. It is unrecognizable when the phase difference δ=0, and the contrast becomes larger with the increase of δ, and the phase inversion will appear when δ continues to increase to a certain value. When using a 90% high absorbance value (high contrast) objective lens, this transformation value is about 0.55λ, and when using a 70% standard absorbance value objective lens, it is about 0.33λ. Objectives with higher absorbance values should be used to resolve smaller optical path differences.
