Experiments in the use of phase contrast microscope

Summary

A dark-field microscope allows you to see the external form of a living cell, but not the internal structure. The phase contrast microscope not only visualizes the morphology of living cells, but also the internal structure of the cells and the continuous process of cell division.

Operation method

phase contrast microscopy

Principle

Using dark-field microscopy, one could observe living cells, but could not see the internal structure of the cells. The emergence of phase contrast microscopy in the 1940s, however, made it possible not only to observe the morphology of living cells, but also to see the internal structure of cells and their processes over time, for which F. Zemike was awarded the 1953 Nobel Prize in Physics. When light passes through a relatively transparent specimen, there is no obvious change in the wavelength (color) and amplitude (brightness) of the light, therefore, it is often difficult to distinguish the morphology and internal structure of unstained specimens (e.g., living cells) when presenting them through an ordinary light microscope, however, due to the difference in the refractive index and thickness of the cellular parts, the light passes through such a specimen, and the light range of the direct light and the diffracted light will be different. As the light range increases or decreases, the phase of the speeded-up or lagging light waves changes (creating a phase difference). The phase difference of light can not be felt by the human eye, but the phase contrast microscope can use the interference phenomenon of light through its special device - ring diaphragm and phase plate, the phase difference of light will be changed into the difference of vibration bats (light and dark difference) that can be perceived by the human eye, thus making the original transparent objects show obvious differences between light and dark and the contrast is enhanced, so that we can be more淸楚. This allows us to observe the living cells and some of the fine structures inside the cells, which are invisible or incomprehensible under the ordinary light microscope and dark-field microscope. Fig. 1 Imaging principle of phase contrast microscope and its setup.

Materials and Instruments

Saccharomyces cerevisiae water-impregnated tablets Bacillus subtilis water-impregnated tablets
phase contrast microscope spotter phase contrast objective microscope coaxial adjustment telescope green filter slide coverslip

Move

1. Replace the microscope's condenser and catch lens with a phase contrast condenser and phase contrast objective, and add a green filter to the light path.
2. The spotlight dial is set to "0" and the light source is adjusted to even out the brightness of the field of view.
3. Place a Brewer's yeast water-soaked slice or a Bacillus subtilis water-soaked slice on a carrier stage, and use a low-power objective (10X) to match light and focus the sample in the bright field.
Phase contrast microscope microscopy on the slide, coverslip requirements are very away, the thickness of the slide should be about 1.0 mm. If it is too thick, the bright ring of the annular diaphragm will become larger, while if it is too thin, the bright ring will become smaller; uneven thickness of the slide, unevenness, or scratches, dust, etc. will also affect the image quality. The standard thickness of coverslip is usually 0.16-10.17 mm, too thin or too thick will make the aberration and color difference increase, affecting the observation results.
4. Set the spotter dial scale to "10" (to match the 10x objective used), and note that when switching from a bright field of view to a ring diaphragm, the aperture of the spotter should be opened sufficiently to increase the brightness of the field of view due to the decrease in the amount of incoming light.
5. Remove the eyepiece and replace it with a coaxial telescope. Fix the outer barrel of the telescope with your left finger, and while observing, turn its inner barrel with your right hand to raise and lower it, focusing so that the bright ring in the spotter and the dark ring in the objective are clear; when the two rings are separated, it means that they are not in-axial, and you can use the center adjusting screw of the spotter to move the bright ring until the two rings are completely rejoined (Fig. 3, B).
6. Perform the co-axial adjustment of the other magnification objectives and the corresponding bad diaphragms in sequence as above.
Accurate co-axial adjustment is the key to achieve good observation results, if the ring of the ring diaphragm and the phase ring in the phase plug objective lens can not accurately match will cause direct light and the light path around the light vein disorder, should be absorbed light can not be absorbed, the delayed phase of the light wave can not be vertically delayed, the loss of the effect of the phase contrast microscope.
7. Remove the binoculars, replace the eyepiece, and use an objective lens of appropriate magnification for observation.

Common Problems

I. Structure of phase contrast microscope

Phase contrast microscope and ordinary optical microscope in the construction of the difference lies in the use of annular diaphragm instead of variable diaphragm, with the phase contrast objective lens instead of the ordinary objective lens, and the need to use the coaxial adjustment of the telescope to straighten the optical axis and the use of color filters.

1. Ring diaphragm

The concentrator diaphragm of a phase contrast microscope is an annular diaphragm, and the illumination light can only burst from the transparent area of the annulus into the concentrator and then be directed obliquely to the specimen (the angle of obliquity is much smaller than that of the dark-field concentrator), which produces both direct light and circular light. Different sizes of ring-shaped diaphragm with a condenser mirror together to form a carousel concentrator, the front of the carousel is marked with a hole, indicating that is located under the condenser mirror diaphragm type, different diaphragms should be used with their respective different magnification of the objective lenses supporting the use of. For example, the symbol of the marked hole is "10", which means that it should be matched with the 10 × objective lens; the symbol is "0", which is the bright field of view of the non-phase-contrast through the light hole (Figure 2A).

Fig. 2 Ring diaphragm (A) and co-axial adjustment telescope (B) for phase contrast microscopy

2. Phase contrast objective lens (lens labeled PC or PH)

The back focal plane of the objective lens is equipped with a phase plate, which is the main device of the phase contrast microscope. The phase plate consists of two parts: a ring-shaped conjugate surface corresponding to the ring-shaped diaphragm and a compensation surface (the inner and outer parts of the conjugate surface), which transmits direct light and transmitted light, respectively. Through the absorption film and delayed phase film coated on the phase plate, the direct light and the winding light will occur light intensity reduction and corresponding changes, and then through the interference of the two, the phase difference into the amplitude difference. 如果是部分吸收直射光而推迟绕射光的相位可产生暗反差（形成明亮的背景和暗的标本），反之，如果是部分吸收绕射光而推迟直射光的相位则产生明反差（标本是明亮的而背景是暗的）。

3. Co-axial adjustment of the telescope

Due to the small size of the aperture ring of the annular diaphragm and the phase ring in the aberration objective, special low magnification telescopes must be used in the co-axial adjustment (Fig. 2, B) to ensure that the apertures of the two rings coincide with each other and that the optical axes are perfectly aligned (Fig. 3).

Figure 3 Phase contrast microscope illumination co-axial adjustment
A: the bright ring formed by the annular diaphragm is smaller than the dark ring on the phase plate, B: correct illumination, the bright ring coincides with the dark ring, C: the center of the annular diaphragm is off-axis.

4. Filter

Green filters are generally used for phase contrast microscopy. This is because the phase contrast objective is mostly achromatic, which only corrects the spherical aberration of yellow and green light but not the spherical aberration of red and blue light, and the green filter is the most effective. In addition, the green filter has a heat-absorbing effect (absorbing red light and blue light), live observation is more favorable.

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