What is a phase contrast microscope? What are the principles and structural characteristics?
What is a phase contrast microscope?
(1) Phase difference microscope principle and structural characteristics
Light waves have different amplitudes (brightness), wavelength (color), and phase (which can be achieved by fluctuations in light at a certain time). When light passes through an object, such as changes in wavelength and amplitude, it can be observed by people's eyes. This is the reason why a stained specimen can be observed under a normal microscope. In living cells and unstained biological specimens, the refractive index and thickness of the micro-structures of the cells are slightly different. When the light waves pass, the wavelength and amplitude do not change, and only the phase changes (the corresponding difference is the phase difference). And this small change can not be identified by the human eye, so it is difficult to observe under ordinary microscope. It is possible to change the phase of direct light or diffracted light, and to use the diffraction and interference phenomena of light to change the phase difference into an amplitude difference (light and dark difference), and it also absorbs part of the direct light to increase the contrast of light and dark. It can therefore be used to observe live cells or unstained specimens. The main difference between the phase contrast microscope and the ordinary microscope is that the annular diaphragm is used instead of the iris diaphragm, and the objective lens with the phase plate (usually marked with PH) is used instead of the ordinary objective lens, and a telescope for the shaft is used. . An annular diaphragm is a diaphragm formed by annular holes of different sizes, and their diameters and hole widths are matched with different objective lenses. Its role is to separate the image formed by direct light from some of the diffractive side images. The phase plate is mounted at the back focal plane of the objective lens, which is provided with an absorption film that absorbs light and a phase film that retards the phase. In addition to delaying the phase of direct or diffracted light, it also absorbs light to change the brightness. The pivoting telescope is used for axis adjustment. When the phase contrast microscope is used, the center of the annular aperture below the condenser and the optical axis of the objective lens should be completely in line. It is necessary to adjust the alignment of the bright ring of the aperture and the annular ring of the phase plate to achieve the performance of the phase contrast microscope. Otherwise, the optical path of the direct or diffracted light is disordered, and the light to be absorbed cannot be absorbed. The light wave of the delayed phase cannot be delayed, and the effect of the phase contrast microscope is lost.
(2) Phase difference microscope use method: The phase difference device is an accessory device in the multi-function series microscope, and is used together with a common microscope.
1. The phase difference device is installed and replaced to remove the concentrator used in the ordinary microscope. The annular diaphragm is mounted on the concentrator holder, and the green filter is placed on it. It can absorb red and blue light to make the wavelength range small. The monochromatic light is illuminated and has an endothermic effect, which makes the phase difference observation good. Then unscrew the ordinary objective lens from the converter and replace it with the phase difference objective lens.
2. Focusing Turn on the light source, rotate the concentrator turntable, and align the “o†with the marking hole to make the ordinary concentrator part enter the light path. First use the low-magnitude phase difference objective lens to perform light focusing and focusing according to the ordinary microscope operation method. Rotate the ring diaphragm so that the diameter and hole width of the diaphragm are compatible with the phase difference objective used. For example, when the phase difference objective is 40X, the aperture of the X40 mark hole is applied.
3. The eyepiece is adjusted to pull out the eyepiece, insert the coaxial telescope, and observe the telescope from the inside of the telescope, and fix the outer cylinder with the left hand; while rotating the telescope inner cylinder with the right hand to lower it, when the focus is focused, the annular aperture is bright. The black ring of the ring and the phase plate, at which point the telescope can be fixed. Then raise and lower the concentrator and adjust the spiral underneath to make the size of the bright ring coincide with the black ring. Then adjust the adjustment knob on the ring diaphragm concentrator left and right to make the two rings completely coincide, such as the bright ring is smaller than the black ring. When located on the inside, the concentrator should be lowered to enlarge the bright ring; otherwise, the concentrator should be raised to make the bright ring smaller. If it is still not fully overlapped if it reaches the maximum limit, it may be that the slide is too thick and should be replaced. After the axis adjustment is completed, the telescope is taken out, replaced with the eyepiece, and observed according to the conventional method. When changing the phase difference objective lenses of different magnifications, use a matching ring diaphragm and re-alignment adjustment every time. When using an oil mirror, cedar oil is added to the surface of the lens on the concentrator and the slide.
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