After understanding the names, structures and functions of the main components of the microscope, in order to better utilize the various functions of the microscope, improve the work efficiency, and ensure the best results in microscopic observation and photomicrography, the user must Understand and master the correct debugging methods and methods of use of the microscope. Especially in the new generation of microscopes, it has a variety of functions, and it is possible to perform a variety of microscopic examination methods. It is particularly important to use correct calibration methods and methods of use. The following is an example of the Axioplan Universal Research Microscope, which briefly describes the debugging and use methods. 1. Adjustment of the microscope illumination light path system In order to make the microscope field of view receive uniform and sufficient illumination, the illumination light path system must be adjusted when the microscope is first installed and debugged. This is the correct use of the microscope and is correct. * The important means of results and the most basic requirements. In addition, correctly mastering the adjustment of the illumination light path system is a necessary step after replacing the light source bulb in the microscope process, and is also a necessary means for checking the performance of the microscope from time to time during daily use. The adjustment of the microscope illumination optical path system mainly has the following four items: (1) Preliminary adjustment of the light source lamp room outside the microscope 1 First open the outer casing of the lamp chamber, and press the spring clip to insert the halogen bulb into the socket. When installing, avoid direct contact with the bulb (can be separated by soft cloth or paper), so as to avoid fingerprints and other dirt on the bulb, affecting the bulb. The service life. 2 Place the lamp room on the table. After turning on the power, use a special screwdriver to adjust the focus knob hole of the lamp (labeled “â†â†’†), so that the filament is projected on the wall 1-2m away, and the filament is imaged. To clear; then adjust the height of the lamp to adjust the wire hole (marked with "-" ), so that the position of the filament is appropriate; then adjust the left and right position of the lamp to adjust the screw hole (marked with "-" ), so that the left and right positions of the filament are suitable . (2) The purpose of the inspection and correction of the position of the light source illuminator (filament) in the microscope is to adjust the image end of the illuminant into the field of view of the objective lens, and to ensure that the field of view of the microscope is adequate from the angle of the light source. Uniform lighting is a prerequisite for adjusting the Kühler lighting system. The basic tools needed: equipped with a microscope for the purchase of a microscope. 1 Unplug the frosted glass sleeve in the lamp library and replace the lamp chamber on the microscope; 2 Select 10× objective lens, open the light source program to find the sample and adjust the focus clearly, then switch to the 40× objective lens to adjust the sample. Clear ( 40 × objective lens can see the full picture of the filament); 3 open the aperture stop and field diaphragm of the condenser to the maximum; 4 Unplug one of the eyepieces, replace the centering telescope, grab the white part, and flex the black eyepiece with the other hand to see the filament image in the field of view; 5 If the position of the filament is not suitable, adjust the “──†hole , adjust the filament image in the horizontal direction, adjust the “──†hole, adjust the wire image in the vertical direction until the filament image is adjusted to just fill the objective lens aperture. Light round image 6 After the adjustment is completed, insert the frosted glass sleeve back into the original position, unplug the center telescope, and change back to the eyepiece for the next adjustment. The above adjustment of the illumination light source lamp chamber outside the microscope and the verification of the position of the light source illuminator in the microscope are performed only when the microscope is first installed and debugged and the bulb is replaced, and the microscope cannot be randomly adjusted when used. In case of confusion, you can return to the original state by following the above steps. (3) the right to adjust the lighting Kule (Kohler) system of the microscope proper commissioning, one of the main job is to adjust the illumination optical system, of which the key is to adjust Cooley lighting system. For everyone who uses a microscope, especially for microphotographers, you should have a certain understanding and mastery of the principle and adjustment steps of the Kühler illumination system in order to fully utilize the functions that the microscope should have. The photos that come out can be more consistent and perfect in effect. The principle of the Kühler illumination system is simply that the light emitted from any point on the light source illuminates the field of view of the microscope, and the light emitted by each point on the light source illuminates, in the field of view of the microscope. A very adequate and uniform illumination is achieved. The purpose of adjusting the Kühler illumination system is to achieve uniform and sufficient illumination of the observed field of view to prevent stray light from affecting or interfering with the image system to avoid fogging on the film during photography. High-adjustment of the necessary components of the Kühler lighting system: field diaphragm, concentrating mirror system for axial adjustment. 1 select 10× objective lens and 10× eyepiece; 2 Put the front lens of the concentrating mirror into the optical path, adjust the aperture stop to a moderate position (not too big or small), then raise the concentrating mirror to the top position, and adjust the concentrating mirror turntable to the bright field "J" position; 3 adjust the field diaphragm to a minimum ( 0.1 ); 4 Place the biological sample that has been sealed on the stage, turn on the light source, and adjust the focus clearly; 5 There will be a partially illuminated area or bright spot in the field of view, which is a blurred image of the field of view, in which the details of the sample can be clearly seen; outside it is a darker view, not necessarily See the details of the sample clearly; 6 condensing the condenser slightly downwards, so that the bright spots in the field of view gradually shrink and slowly become a clear polygonal image, which is a clear image of the field of view; 7 In general, the polygon image is not in the center of the field of view. You need to adjust the pair of centering screws of the condenser to adjust the image of the field diaphragm polygon to the center position. 8 gradually open the large field of view, so that the polygon image becomes the inscribed polygon of the field of view, further check the condition of the adjustment, such as the alignment is not ideal, continue to fine-tune the centering screw; 9 The field diaphragm is slightly enlarged slightly, so that its polygon image just disappears on the edge of the field of view. At this point, the Kule illumination system is adjusted. After the adjustment of the Kühler lighting system, the entire field of view is illuminated evenly, and the micrographs taken are bright and clear with normal contrast. In the future use, special attention should be paid to: a. The field diaphragm cannot be opened arbitrarily, but the field diaphragm can be reduced with the increase of the objective magnification, and it will be enlarged with the decrease of the objective magnification; b. Condenser The position of the high and low is not allowed to be adjusted, otherwise it will destroy the adjusted Kühler illumination system; c. When using the objective lens of 10× , the front lens of the condenser should be out of the optical path, and when using the objective lens of 10× or 10× , the front end should be used. The lens is placed in the optical path; d. Regarding the problem of matching the objective lens multiple with the field diaphragm size, in actual use, as a general observation, it is not necessary to receive a small or open field diaphragm, but for photomicrography, To avoid the interference of stray light on the camera system, so that you can take a more perfect picture, you should adjust the field diaphragm to just disappear on the side of the observed field of view when using each objective lens. More complicated work, but it is not impossible. A relatively simple method is to adjust the field diaphragm corresponding to each of the multiple objective lenses in advance, and make a mark, and then directly adjust to the corresponding position according to the mark when using. (4) Correct use of the aperture stop Since the aperture stop of the condenser can affect the resolution of the microscope, the correct use method should be mastered when using it. In the past, due to insufficient understanding of the aperture stop, it was often used as a tool to adjust the brightness of the field of view. Although adjusting the aperture stop can change the brightness of the field of view to a certain extent, it will directly affect the contrast, contrast and resolution of the image, and should be avoided as much as possible during use. In order to play the role of the concentrating aperture of the concentrating mirror, in order to obtain the best resolution when observing, especially in photomicrography, it is necessary to adjust the aperture stop after the sample is sharply adjusted, when each objective lens is replaced by one. Make it exactly 2/3 of the numerical aperture of the objective lens (the aperture of the objective lens) . The adjustment method is to focus on the black phase difference ring in the field of view with the centering telescope, adjust the aperture stop, and you can see a polygonal aperture stop image, and then adjust to 2/3 of the objective lens aperture image , that is, between the black phase difference rings. Between the outer and the circular view. For the sake of convenience, the aperture stop corresponding to each multiple objective lens can be pre-adjusted and marked so as not to be re-adjusted every time. 2. Microscope imaging optical path system adjustment and microscopic examination summary Microscopic imaging optical path system adjustment is carried out according to the needs of different microscopy procedures. The so-called microscopy (Microscopy), generally for the method is to illuminate the sample when the microscope is used, and how the sample image formed by techniques and methods to obtain a better contrast. The following is a brief description of several methods that have been matured in microscopy and the corresponding adjustment methods for the microscope imaging optical path system. (1) Transmitted bright field ( brightfield ) This is the most traditional and most popular application method since the invention of the microscope. Basic components: a. Objective lens: any objective lens can be used for bright field observation; b. Condenser: all kinds of condensers can be used, preferably with aperture stop. Adjustment method: After the Kule illumination system of the above microscope is adjusted, the bright field method can be applied. Scope of application: All stained tissue sections, blood smears, etc. Matters needing attention: a. When using the bright field method, the Kule illumination system must be adjusted well; b. The field diaphragm cannot be opened arbitrarily. When using the objective lens of 10× , 10× or less and 10× or more, the condenser should be used. The front lens is separately reflected and placed into the optical path; c. The aperture of the concentrating mirror cannot be used to adjust the brightness of the field of view, and the height of the concentrating mirror is not adjusted. Otherwise, the resolution and damage of the microscope are reduced. Culler illumination system; d. For photomicrography, each time you use a multiple of the objective lens, adjust the aperture stop of the condenser so that its size is exactly 2 / 3 of the numerical aperture of the objective lens used . (2) the phase difference of transmitted light method (phase-contrast) This is a modern contrast microscopy enhancement method. Basic components: phase difference objective lens, multi-purpose condenser with bright field and phase difference, centering telescope, green filter. Adjustment method: a. On the basis of the adjustment of the Kühler illumination system, use the bright field method to adjust the focus of the sample clearly; b. Turn the condenser to the position of the Ph1 alignment dial, select the 10× phase difference objective lens, and change it to be observed. transparent sample; wherein an eyepiece pulled C, put on the telescope, and focusing on the difference between the two rings on the field of view (objective lens and the condenser lens ring black phase difference of light transmissive phase difference ring);. D Vision. The two phase difference rings do not necessarily overlap, and the two adjusting devices on the concentrating mirror (adjusting the adjusting rods at the left and right positions of the phase difference ring and the friction type adjusting buttons at the front and rear positions) are adjusted to make the light transmitting ring move back and forth and left and right to coincide with the black ring; e. After adjustment, change back to the observation eyepiece, press the green filter into the optical path, and observe the phase difference image of the sample; f. When there is 20× and 40× objective lens, the condenser should be set at Ph2 position. When using a 100 objective lens, the condenser should be placed at the Ph3 position. Scope of application: Suitable for observing samples that are transparent, unstained or unstainable, such as various cells, living tissue, unstained or unstained tissue sections, aquatic organisms, etc. (3) Differential interference-contrast ( DIC ) In order to overcome the contrast between the sample details when the phase difference method is observed, the details that should be seen are masked, and the thickness of the sample or tissue slice is quite thin. The principle can be thicker than 10?m and other limitations, using the principle of two-beam interference to design the sub-differential interference phase contrast method. The method of adjusting a system must be modulated to Ku Leming adjusted on the basis of a good method of DIC;. B first with 10 × objective lens, in order to determine a good bright field sample can clearly see the position of the objective lens focusing;.. C The The polarizer ( orer ) is placed in the illumination path, and the orientation should be east - west; d. Turn the condenser dial to the position corresponding to the 10× objective, ie DIC 0.3-0.4 ; e. behind the objective or insert 10 × objective lens nosepiece using DIC tabs (DIC slider); f the analyzer (analyzer) is inserted into the imaging optical path, attention to its orientation should South - North;.. g put transparent be observed Sample, open the light source to make the sample focus clear; h. Adjust the DIC insert to make the differential interference phase contrast image achieve the best effect, that is, the relief effect is the most obvious; i. At the same time, the aperture aperture of the condenser can be adjusted to make the contrast The effect is also optimal; j. Then fine-tune the details of the sample to see the structure at different levels in the sample; k. If the first order red retardation plate is inserted and the DIC insert is adjusted at the same time , Seen in the field of vision The changing of brilliant colors, red, orange, yellow, green, blue, purple, pink, purple and gold have. Scope of application: Tissue sections that are transparent or incapable of staining, thickness up to about 100 μm , living tissue and living cells, and small living organisms in culture. (4) Method epifluorescence (incident-loght fluorescence Epi-FL ) referred to as epi-fluorescence excited by light, modern microscopy is newly developed out of a powerful contrast enhancement method. The light source for exciting fluorescence is changed above the objective lens, and the light is incident from the objective lens through the mirror to the objective lens to excite the sample, and the excited fluorescence from the sample is imaged by the objective lens and penetrates the mirror to be observed by the eyepiece. The method is simple and efficient, and the intensity of the 50W light source is stronger than the 250W of the transmission fluorescence method . The fluorescence method uses short-wavelength ultraviolet light, violet light, blue-violet light, blue light, and green light to excite the sample. As long as the sample contains a fluorescent component, it absorbs the short-wave excitation light and emits a longer-wavelength fluorescence. . Different substances can only absorb the excitation light of a specific wavelength, and the released fluorescence also has a specific wavelength, so it is very effective for specific identification, such as some pathogenic bacteria and spirals, which can be emitted by ultraviolet light. Their unique fluorescence is easy to identify. This method of using the substance to absorb the excitation light and emit the characteristic fluorescence is called autofluorescence. Some substances do not absorb the excitation light themselves, or can not release fluorescence after absorption, but can absorb or adsorb specific fluorescent pigments or dyes, and these specific fluorescent pigments or dyes can only absorb specific excitation light, and then release specific The fluorescence, which indirectly identifies a substance, is called indirect fluorescence. The above fluorescence methods are widely used in the specific research and identification of medicine, biology and industry. Adjustment method: Fluorescence microscope or microscope with fluorescent parts, the adjustment method is roughly the same. 1 Installation of mercury lamp a. Open the package, remove the mercury lamp and carefully install it on the upper electrode cooling cap. When installing, pay attention to the finger can not directly touch the front of the lamp tube and the heat-dissipating cap. The sealing port of the mercury lamp should be opposite to the heat-dissipating cap. Left or right side; b. Install and fix the upper electrode of the mercury lamp on the small hole on the bottom surface of the heat dissipation cap, and then install the lower electrode of the mercury lamp and the other end of the upper electrode lead on the lamp holder respectively. Fix the hole in the jack; c. After locking the screw on the heat sink, carefully insert the mercury lamp into the lamp room together with the lamp holder and the heat dissipation cap, lock the corresponding screws, and then plug the cable and socket on the lamp holder. Mercury lamp power supply on the dedicated socket at the rear. d. For detailed installation methods, please refer to the relevant instructions. 2 preliminary adjustment of the mercury lamp lamp room outside the microscope: a. Turn on the mercury lamp power supply and let the mercury lamp preheat for 10-15min ; b. Place the mercury lamp lamp room on the table and let the mercury arc project to 2-3m On the outer wall, draw a horizontal line with the same height as the center line of the lamp room window as the reference line; c. Turn the lamp room focus knob to make the image of the mercury arc clearly projected onto the wall; d. Adjust the lamp room separately adjusting screw holes 5, the mercury arc image into a reflected image side by side and nearly * possible, but do not overlap on the housing. 3 Mercury lamp mercury arc inspection in the microscope position: a. The field light in the mercury lamp illumination light path system is opened to the maximum; b. Push the fluorescence filter group to the position where the blue light is excited to avoid the mercury lamp The blue light in the middle is too glaring; c. Place the observed sample or a glass slide on the object table, and cover a thin paper that is slightly larger and whiter than the cover glass; d. Remove the objective lens to make the excited blue light The space of the objective lens converter is illuminated on white paper. There will be a blue circular illumination area on the white paper. The image of the mercury arc and its reflection image should appear in the central part of the area. Otherwise, the lamp room can be adjusted. Adjust the focus knob to the clearest, then adjust the five adjustment screw holes on the lamp housing separately until the image of the mercury arc and its reflection image are placed side by side in the center of the illumination area. e. After adjustment, put the objective lens back, through the eyepiece to see the yellow-green fluorescence emitted by the white paper after being excited by blue light; f. Carefully adjust the focus to see the fibers of the white paper; remove the white paper, the fluorescent details on the sample It is faintly visible and easy to focus. 4 mercury lamp use precautions Currently used is a 50W ultra-high pressure mercury lamp, the lamp tube is connected with a pair of tungsten electrode and liquid mercury (attached to the pipe wall at room temperature), when not ignited, the pipe pressure is very low, in the lamp After the voltage is applied between the two electrodes of the tube, the mercury is vaporized into mercury vapor to form a mercury arc to generate strong light, and the temperature rises, and the gas pressure in the tube rapidly rises to 10 atmospheres. Because of the high-pressure gas discharge, it is necessary to understand its characteristics in order to safely use the mercury lamp. a. After the mercury lamp is turned on, it takes 10-15 minutes to warm up, and the mercury can be fully vaporized and form a mercury arc, which produces high-intensity and stable excitation light. Therefore, it is necessary to energize before the observation; b. During the use of the mercury lamp, do not arbitrarily switch the power of the mercury lamp; c. After turning off the power of the mercury lamp, it must wait for 15-20min , and then wait for the mercury lamp to cool after self-ignition. Passing the power supply, in violation of this operation, will have serious consequences! Since the mercury vapor in the mercury lamp is not completely liquefied, the internal resistance of the mercury vapor is very small. Once the voltage is applied between the two electrodes by energization, a strong current is formed in the mercury lamp, and the choke coil in the power source of the mercury lamp is burned off. In the case of mercury lamps, mercury vapors pervaded the entire laboratory, causing poisoning of workers, not only the loss of mercury lamps, but also the collection of light - concentrating components in the lamp chamber . d. The service life of mercury lamp is generally only 300h , and it can be used up to 600h . The service life is inversely proportional to the number of switches. It should be observed in a batch of samples for 2-3h . Mercury lamps are expensive and should be cherished. e. The end of the life of the mercury lamp is marked by the difficulty of ignition and the blackening of the lamp. 5. Dark field Many transparent or translucent samples, such as bacteria, microorganisms, fine structures in cells, and crystal inclusions, are not easily seen in bright-field microscopy, if dark-field methods are used. It can greatly improve the visibility of the sample. What is seen in the dark field method is the outline of the sample and its details that illuminate in the dark field of view. The maximum resolution of a general optical microscope is 0.2 μm . Although the dark field microscope does not clearly distinguish the detailed structure of the sample, it can see the presence of fine particles above 0.004 μm , that is, the submicroscopic structure can be seen. Suitable for observing fine particles and bacteria. Adjustment method: The main necessary component of the dark field method is the dark field condenser. The Kule illumination system must be adjusted with a bright field condenser before use. When changing to the dark field condenser, remove the slide (sample), place the immersion oil on top of the condenser, and place the sample slide on the table. The immersion oil is filled between the two. The condenser must be used with a 100x oil mirror fitted with an iris diaphragm . Another medium-magnification dry dark field concentrating mirror can be used with a medium-magnification objective lens. The concentrating mirror has a central light block, and the illumination light can only enter the concentrating mirror through the light-transmitting ring between the optical file and the edge of the concentrating mirror. For a fully equipped phase contrast microscope, the phase difference concentrating ring with the Ph3 objective lens can be used with a phase difference objective of 10× and 10× to form a low-magnification dark field effect. Rongcheng Jingyi Oceanic Technology Company Limited , https://www.jingyifoods.cn