Joke Collection Website - Bulletin headlines - Questions about using the microscope

Questions about using the microscope

1. Operating steps and precautions

(1) Upright microscope

1. Placement

Hold the mirror arm with your right hand. Hold the mirror base with your left hand to keep the mirror body upright. The desktop should be clean and stable, and should be placed near a window or in a well-lit place. The single tube is usually placed on the left side, 3 to 4 cm away from the edge of the table.

2. Cleaning

Check whether there is any problem with the microscope and whether it is clean. The mechanical part of the mirror body can be wiped with a clean soft cloth. The lens should be wiped with lens cleaning paper. If there is glue or dirt, use a small amount of xylene to clean it.

3. Align the light

Raise the lens tube to a distance of 1 to 2 cm from the stage, and align the low-power lens with the clear aperture. Adjust the aperture and reflector. Use a flat mirror when the light is strong, and a concave mirror when the light is weak. Use both hands to turn the reflector.

If you are using a microscope with a light source, you can omit this step, but you will need a knob to adjust the brightness.

4. Install the specimen

Place the slide on the stage, making sure the side with the cover slip is facing up. Fix the glass slide with a spring clip and turn the knob of the platform mover to align the material to be observed with the center of the light hole.

5. Focusing

When adjusting the focus, first rotate the coarse focusing knob to slowly lower the lens barrel, and observe carefully from the side until the objective lens is close to the slide specimen, and then use the left eye Observe from the eyepiece, turn the coarse focus knob with your left hand to raise the lens barrel until you can see the specimen image clearly, and then use the fine focus knob to adjust it back to clarity.

Operation note: Do not adjust the focus directly under high magnification; when the lens tube is lowered, observe the distance between the lens tube and the specimen from the side; you must understand the critical value of the object distance.

If using a binocular microscope, if there is a difference in visual acuity between the two eyes of the observer, the visual acuity adjustment ring can be used to adjust it. In addition, the binoculars can be moved relative to each other to adapt to the distance between the operator's eyes.

6. Observation

If you use a monocular microscope, open your eyes naturally, observe the specimen with your left eye, observe, record and draw with your right eye, and adjust the focus with your left hand to make the object image clear and clear. Move the specimen field of view. The right hand records and draws.

During microscopic examination, the specimen should be moved in a certain direction and the field of view should be moved until the entire specimen is observed so that no inspection is missed or repeated.

Adjustment of light intensity: Under normal circumstances, the light for dyed specimens should be strong, and the light for colorless or unstained specimens should be weak; the light for low-power observation should be weak, and the light for high-power observation should be strong. In addition to adjusting the reflector or light source, the adjustment of the iridescent aperture is also very important.

1) Low-power lens observation

When observing any specimen, you must first use a low-power lens because its field of view is large and it is easy to find the target and determine the part to be observed.

(2) High-magnification lens observation

When switching from low-magnification lens to high-magnification lens, you only need to slightly adjust the fine focus knob to make the object image clear.

Do not use the coarse focus knob when using a high-magnification lens, otherwise the cover glass may be crushed and the lens may be damaged.

When turning the nosepiece converter, do not use your fingers to push the objective lens directly. This will easily cause the optical axis of the objective lens to deflect, and the threads of the converter will be damaged due to uneven stress, which will eventually lead to the converter being scrapped.

(3) Observation of the oil lens

Use a low-power lens and a high-power lens to move the object to be inspected to the center of the field of view, and then change the oil lens for observation. Before observing with an oil lens, the brightness of the microscope should be adjusted to the brightest setting and the aperture should be fully opened.

When using an oil lens, first add a drop of cedar oil (mirror oil) on the cover slip, then lower the lens barrel and observe carefully from the side until the oil lens is immersed in the cedar oil and close to the specimen, and then Observe with the eyepiece, and use the fine focus knob to raise the lens barrel until you can see the focal length of the specimen clearly, stop and adjust it for clarity.

The appropriate amount of cedar oil should be added. After using the oil lens, be sure to use lens cleaning paper dipped in xylene to wipe off the cedar oil, and then use dry lens cleaning paper to wipe off excess xylene.

7. End the operation

After the observation is completed, remove the sample, rotate the converter so that the lens is V-shaped on both sides, the reflector should be upright, lower the lens barrel, and wipe it clean. , and put on the lens cover.

If you are using a microscope with a light source, you need to adjust the brightness knob to the darkest light, and then turn off the power button to prevent excessive current from burning out the light source the next time you turn it on.

Generally, when using a microscope, the objective lens and the slide should be closer together. I don’t know the specifics. But do not make contact, then use the coarse adjustment screw to adjust it upward

Reference: /question/70192366.html

Share it with your friends:

i Tieba

Sina Weibo

Tencent Weibo

QQ Zone

Renren

Douban

MSN

Helpful to me

26 Answer time: 2009-3-6 22:04 | Let me comment | Report

To TA asked for help Respondent: Kekezhou | Level 2

Area of ??Expertise: Not customized yet

Activities participated in: No activities participated in yet

Asker's answer Rating:

Thank you! !

Other answers*** 1

1. Adjustment of the microscope illumination light path system:

In order to ensure that the field of view of the microscope can be uniformly affected And for sufficient lighting, when the microscope is first installed and debugged, the lighting light path system must be adjusted. This is an important means and the most basic requirement for using the microscope correctly and obtaining correct and reliable results. In addition, correctly mastering the adjustment of the illumination light path system is a necessary step after replacing the light source bulb during use of the microscope. It is also a necessary means to test the performance of the microscope from time to time during daily use. The adjustment of the microscope illumination light path system mainly includes the following four items:

1. Preliminary adjustment of the illumination light source lamp house outside the microscope

① First, open the shell of the lamp house and press The spring clip installs the halogen bulb into the socket. When installing, avoid direct contact with the bulb with your fingers (can be separated by soft cloth or paper) to avoid fingerprints and other dirt on the bulb, which will affect the service life of the bulb.

② Place the lamp house on the table. After turning on the power, use a special screwdriver to adjust the focusing knob hole of the lamp (marked "←→") so that the filament is projected 1-2m away. On the wall, adjust the filament image to be clear; then adjust the height adjustment screw hole (marked with "——") of the lamp to make the filament position appropriate; then adjust the left and right position adjustment screw holes of the lamp (marked with "——" ) to make the left and right positions of the filament appropriate.

2. The purpose of checking and correcting the position of the light source illuminator (filament) in the microscope is to adjust the image end of the illuminant correctly into the field of view of the objective lens, and to ensure that it is correct from the perspective of the light source. The field of view of the microscope is fully and uniformly illuminated, which is a prerequisite for adjusting the Kuhler illumination system. Basic tools needed: Centering the telescope is already equipped when purchasing the microscope.

① Unplug the ground glass sleeve in the lamp library and put the lamp house back on the microscope;

② Use the 10× objective lens, turn on the light source program to find the sample and adjust the focus clearly. Then switch to a 40× objective lens to focus the sample clearly (the 40× objective lens can see the entire filament clearly);

③ Open both the aperture diaphragm and the field diaphragm of the condenser to the maximum;

④ Unplug one of the eyepieces, replace it with the centering telescope, grab the white part, and stretch the black eyepiece with the other hand, you can see the filament image in the field of view;

⑤ If the filament position If it is not suitable, adjust the "——" hole and adjust the filament image in the horizontal direction. Adjust the "——" hole and adjust the filament image in the vertical direction until the filament image is adjusted to a circular light image that just fills the objective lens aperture;

⑥ After the adjustment is completed, insert the ground glass sleeve back into its original position, unplug the centering telescope, and replace the eyepiece for the next adjustment. The above-mentioned adjustment of the lighting source lamphouse outside the microscope and the calibration of the position of the light source illuminator within the microscope only need to be carried out when the microscope is first installed and debugged and the bulb is replaced. Do not make random adjustments when using the microscope. In case of chaos, you can follow the above steps to restore it to its original state.

3. Correct adjustment of the Kohler illumination system. One of the main tasks for correct debugging of the microscope is the adjustment of the illumination light path system, and the key is the adjustment of the Kohler illumination system. For everyone who uses a microscope, especially those who take microphotographs, they should have a certain understanding and mastery of the principles of the Kuhler illumination system and its adjustment steps, so that they can fully utilize the functions of the microscope and take photos. Only then can the photos produced be more consistent and perfect in effect. Simply put, the principle of the Kuhler illumination system is: the light emitted from any point on the light source illuminator can illuminate the field of view of the microscope, and the light emitted from each point on the light source illuminator is gathered together and illuminates the field of view of the microscope. Achieving very sufficient and even illumination. The purpose of adjusting the Kuhler illumination system is to obtain uniform and sufficient illumination of the observed field of view, to prevent stray light from affecting or interfering with the imaging system, and to prevent fog from forming on the film during photography. Necessary components of the highly adjustable Kuhler illumination system: field diaphragm, condenser system with on-axis adjustment.

① Select 10× objective lens and 10× eyepiece;

② Place the front lens of the condenser into the light path, and adjust the aperture diaphragm to a moderate position (not too big, not too small), Then raise the condenser to the top position, and adjust the condenser dial to the bright field "J" position;

③ Adjust the field diaphragm to the minimum (0.1);

④ Place the sealed biological sample 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 the function of the field diaphragm. Blurred image, in which the details of the sample can be clearly seen; outside it is a darker field of view, and the details of the sample may not be clearly visible;

⑥ Adjust the condenser downward slightly , so that the bright spots in the field of view gradually shrink and slowly turn into a clear polygonal image, which is the clear image of the field diaphragm;

⑦ Generally, the polygonal image is not in the field of view In the center, you need to adjust a pair of centering screws of the condenser to adjust the polygonal image of the field diaphragm to the central position;

⑧ Gradually open the field diaphragm to make the polygonal image become an inscribed polygon of the field of view. , further check the centering situation. If the centering is not ideal, continue to fine-tune the centering screw;

⑨ Open the field diaphragm slightly and then slightly wider, so that its polygonal image just disappears in the field of view. On the edge of the domain, at this point, the Kuhler lighting system has been adjusted. After the Kuhler lighting system is adjusted, the entire field of view is evenly illuminated, and the photomicrographs taken are bright and clear with normal contrast.

Special attention should be paid to the following during future use: a. The field diaphragm cannot be opened arbitrarily, but it can be narrowed as the objective lens magnification increases, and can be opened as the objective lens magnification decreases; b. Condenser lens Do not adjust the height of the condenser arbitrarily, otherwise the adjusted Kuhler illumination system will be destroyed; c. When using an objective lens below 10×, the front lens of the condenser must be placed out of the optical path. When using an objective lens of 10× or above, the front lens must be moved out of the optical path. Put the lens into the optical path; d. Regarding the coordination between the objective lens magnification and the field diaphragm size, in actual use, it is not necessary to close or widen the field diaphragm for general observation, but when doing microphotography, in order to To prevent stray light from interfering with the photographic system, in order to take better pictures, when using an objective lens of each magnification, the field diaphragm should be adjusted to just disappear at the edge of the field of view being observed. This is A relatively complicated job, but it must be done. A simpler method is to adjust the field diaphragm corresponding to each magnification objective lens in advance and mark it, and then directly adjust it to the corresponding position according to the mark when using it later.

4. Correct use of the aperture diaphragm. Since the aperture diaphragm of the condenser can affect the resolution of the microscope, you should master the correct usage method when using it. In the past, due to insufficient understanding of the aperture diaphragm, it was often regarded as a tool for adjusting the brightness of the field of view. Although adjusting the aperture diaphragm can change the brightness of the field of view to a certain extent, it will directly affect the contrast, contrast and resolution of imaging, and should be avoided as much as possible during use. In order to give full play to the role of the condenser aperture diaphragm and obtain the best resolution when observing, especially when taking microphotography, it is necessary to adjust the aperture diaphragm after each objective lens is changed to a magnification and after the sample is focused clearly. Make its size exactly equal to 2/3 of the numerical aperture (objective lens aperture image) of the objective lens used. The adjustment method is to use the centering telescope to focus on the black phase contrast ring in the field of view, adjust the aperture diaphragm, you can see a polygonal aperture diaphragm image, and then adjust it to be equal to 2/3 of the objective lens aperture image, that is, between the black phase contrast ring Between the outside and the inside of the circular field of view. For convenience, the aperture diaphragm corresponding to each magnification objective lens can be pre-adjusted and marked to avoid having to readjust it every time it is used.

2. Adjustment of the microscope imaging light path system and overview of microscopy techniques:

The adjustment of the microscope imaging light path system is based on the needs of different microscopy techniques. The so-called microscopy, in a nutshell, refers to the lighting method used when observing samples with a microscope, as well as the techniques and methods of how to obtain better contrast in the images formed by the samples. The following is a brief description of several mature methods in microscopy and the corresponding adjustment methods of the microscope imaging light path system.

1. Transmitted light field of view:

This is the most traditional and common 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 lens: All kinds of condenser lenses are available, preferably equipped with an aperture diaphragm. Adjustment method: After the Kuhler 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. Notes: a. When using the brightfield method for observation, the Kuhler illumination system must be adjusted; b. The field diaphragm cannot be opened arbitrarily. When using objective lenses of 10×, 10× below and 10× above, the condenser must be adjusted. The front lens is moved out and into the light path respectively; c. Do not use the aperture diaphragm of the condenser to adjust the brightness of the field of view, and do not adjust the height position of the condenser randomly, otherwise it will reduce the resolution of the microscope and destroy the adjusted Kuhler illumination system; d. When doing microphotography, every time you use an objective lens of another magnification, you must adjust the aperture diaphragm of the condenser so that its size is exactly equal to 2/3 of the numerical aperture of the objective lens used.

2. Transmitted light phase contrast method:

This is a contrast enhancement method in modern microscopy. Basic components: phase contrast objective lens, multi-purpose condenser for both bright field and phase contrast, centering telescope, and green filter.

Adjustment method:

a. Based on the adjustment of the Kuhler lighting system, use the bright field method to focus the sample clearly;

b. Turn the condenser to Ph1 and align it with the dial mark, select a 10× phase contrast objective, and replace it with the transparent sample to be observed;

c. Unplug one of the eyepieces, replace it with the centering telescope, and adjust the focus to On the two phase contrast rings in the field of view (the black phase contrast ring of the objective lens and the light-transmitting phase contrast ring of the condenser);

d. The two phase contrast rings in the field of view may not coincide with each other. Adjust the two adjustments on the condenser. device (the adjusting lever for adjusting the left and right position of the phase difference ring and the friction knob for adjusting the front and back position), so that the light-transmitting ring moves forward, left, and right and coincides with the black ring;

e. After adjustment, change back Use the eyepiece for observation and press the green filter into the light path to observe the phase difference image of the sample;

f. When observing with 20× and 40× objective lenses, the condenser should be set at the Ph2 position. When using a 100 objective lens, the condenser should be set to the Ph3 position.

Scope of application: Suitable for observing transparent, unstained or unstainable samples, such as various cells, living tissues, unstained or unstained tissue sections, aquatic organisms, etc.

3. Differential interference contrast method:

In order to overcome the halo surrounding the sample details when observing with the phase contrast method, it will obscure the details that should be seen, as well as the sample or The thickness of tissue slices is required to be quite thin. In principle, it can be thicker than 10μm and other limitations. The sub-differential interference contrast method is designed using the principle of double-beam interference.

Adjustment method:

a. The DIC method must be adjusted after the Kulemin system has been adjusted;

b. First use 10× Objective lens, use bright field to determine the focusing position of the objective lens that can clearly see the sample;

c. Place the polarizer into the illumination light path, and note that its orientation should be east-west. ;

d. Turn the condenser turntable to the position corresponding to the 10× objective lens, that is, DIC 0.3-0.4;

e. Insert the 10× objective lens behind the objective lens or on the objective converter. ×The DIC slider used in the objective lens;

f. Insert the analyzer into the imaging optical path, and note that its orientation should be south-north;

g . Replace the transparent sample to be observed, turn on the light source and focus the sample clearly;

h. Adjust the DIC insert to achieve the best effect of the differential interference contrast image, that is, the most obvious relief effect ;

i. At the same time, the aperture diaphragm of the condenser can be adjusted to achieve the best contrast effect;

j. Then fine-tune the details of the sample to see different layers of the sample The structure on;

k. If you insert the color complementer (first order red retardation plate) and adjust the DIC insert at the same time, you can see the constantly changing brilliant colors in the field of vision, red, orange, yellow There are , green, blue, purple, pink, pink purple and golden yellow. Scope of application: Transparent or non-stainable tissue sections with a thickness of up to about 100?m, living tissues and cells in culture, microorganisms, etc.

4. Incident-loght fluorescence Epi-FL:

Referred to as epi-fluorescence, it is a newly developed method in modern microscopy. Powerful contrast enhancement method. It changes the light source used to excite fluorescence above the objective lens. The light is injected from above the objective lens through the reflector into the objective lens to excite the sample. The fluorescence excited from the sample is imaged by the objective lens and penetrates the reflector to be observed by the eyepiece. This method is simple and efficient, and the light source intensity of 50W is stronger than the 250W of the transmitted 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 components that can produce fluorescence, it will absorb the short-wavelength excitation light and release longer-wavelength fluorescence. .

Different substances can only absorb excitation light of specific wavelengths, and the fluorescence released will also have specific wavelengths, so it is very effective for specific identification. For example, some pathogenic bacteria and spirochetes can emit light after being excited by ultraviolet light. Their unique fluorescence can be easily identified. This method of using substances to absorb excitation light and then emit unique fluorescence is called autofluorescence. Some substances themselves do not absorb the excitation light, or cannot release fluorescence after absorbing it, but they 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 fluorescence. The fluorescence can indirectly identify a certain substance, which is called the indirect fluorescence method. The above fluorescence methods are widely used in specific research and identification in medicine, biology and industry. Adjustment method: The adjustment methods for fluorescence microscopes or microscopes with fluorescent components are roughly the same.

① Installation of mercury lamp:

a. Open the package, take out the mercury lamp and carefully install it on the upper electrode heat dissipation cap. When installing, be careful not to let your fingers directly touch the lamp tube and heat dissipation. On the front of the cap, the air sealing port of the mercury lamp should face the left or right side of the heat dissipation cap;

b. Install and fix the upper electrode of the mercury lamp to the small hole on the bottom of the heat dissipation cap. Then install the lower electrode and the other end of the upper electrode lead of the mercury lamp into their respective sockets on the lamp holder and fix them;

c. After tightening the screws on the heat sink, attach the mercury lamp together with the lamp. Carefully install the holder and heat dissipation cap into the lamp chamber, tighten the corresponding screws, and then insert the connection wires and sockets on the lamp holder into the dedicated socket at the rear of the mercury lamp power supply.

d. Please refer to the relevant instructions for detailed installation methods.

② Preliminary adjustment of the mercury lamp lamp chamber outside the microscope:

a. Turn on the power of the mercury lamp and let the mercury lamp preheat for 10-15 minutes;

b. Place the mercury lamp chamber on the table, let the mercury arc project onto the wall 2-3m away, and draw a horizontal line that is the same height as the center line of the lamp chamber window to the ground as a reference line;

c. Turn the focusing knob of the lamp house so that the image of the mercury arc is clearly projected on the wall;

d. Adjust the 5 adjusting screw holes on the lamp house casing respectively to adjust the image of the mercury arc to its other parts. The reflected images are adjusted to be side by side and as close as possible, but do not overlap.

③ Inspection of the position of the mercury arc of the mercury lamp in the microscope:

a. Open the field diaphragm in the mercury lamp illumination light path system to the maximum;

b. Push the fluorescence filter set to the blue light excitation position to avoid the blue light in the mercury lamp being too dazzling;

c. Place the observed sample or a slide on the object stage , cover it with a piece of white tissue paper that is slightly larger than the cover glass;

d. Remove an objective lens, let the excited blue light shine on the white paper through the gap of the objective lens converter, and the white paper There will be a blue circular lighting area. The image of the mercury arc and its reflected image should appear in the center of this area. Otherwise, you can adjust the focusing knob of the lamp house to the clearest, and then adjust the 5 adjustment screw holes until the image of the mercury arc and its reflected image are aligned side by side in the center of the lighting area.

e. After adjustment, put the objective lens back, and the yellow-green fluorescence emitted by the white paper after being excited by blue light can be seen through the eyepiece;

f. Carefully adjust the focus to see clearly The fibers of the paper; remove the white paper, and the fluorescent details on the sample are faintly visible and can be easily focused.

④ Precautions for the use of mercury lamps. Currently, the 50W ultra-high pressure mercury lamp is commonly used. The lamp tube has a pair of tungsten electrodes and liquid mercury (attached to the tube wall at room temperature). When not ignited, The air pressure in the tube is very low. After a voltage angle is applied between the two electrodes of the lamp to ignite, the mercury vaporizes into mercury vapor to form a mercury arc and generates strong light. As the temperature rises, the air pressure in the tube quickly rises to 10 atmospheres. Since it is a high-pressure gas discharge, its characteristics must be understood in order to use mercury lamps safely.

a. After the mercury lamp is powered on, it requires 10-15 minutes of preheating time before the mercury can fully vaporize and form a mercury arc, producing high-brightness and stable excitation light.

Therefore, it is necessary to turn on the power in advance before observation;

b. When using the mercury lamp, do not turn on and off the power of the mercury lamp at will;

c. After turning off the power of the mercury lamp, you must wait 15-20 minutes, wait for the mercury lamp to self-ignite and cool down before turning on the power again. Violation of this operating regulation will cause 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 electricity is applied and a voltage is applied between the two electrodes, a strong current will form in the mercury lamp, which may blow out the fuse or burn out the choke in the mercury lamp power supply. , or worse, the mercury lamp exploded, and mercury vapor filled the entire laboratory, causing the staff to be poisoned. Not only did they lose the mercury lamp, but they also blew up the light-concentrating components in the lamp room.

d. The service life of mercury lamps is generally only 300 hours, and can reach 600 hours if used properly. The service life is inversely proportional to the number of switches. A batch of samples should be concentrated for 2-3 hours of observation. Mercury lamps are extremely expensive and should be used with care.

e. The end of the life of a mercury lamp is marked by difficulty in ignition and blackening of the lamp tube.

5 Dark field method (dark field):

Many transparent or translucent samples, such as bacteria, microorganisms, fine structures within cells and the contents of crystals, etc., can be detected in It is difficult to see clearly in a bright field microscope. If the dark field method is used, the visibility of the sample can be greatly improved. What you see with the dark field method is the outline of the sample and its details that glow against the dark field background. The highest resolution of a common optical microscope is 0.2μm. Although a dark-field microscope cannot clearly distinguish the detailed structure of a sample, it can see the existence of fine particles above 0.004μm, that is, it can see submicroscopic structures, especially Suitable for observing fine particles and bacteria. Adjustment method: The main necessary component of the darkfield method is the darkfield condenser. Before use, the Kuhler illumination system must be adjusted with a bright field condenser. When replacing the dark field condenser, you need to remove the slide (sample), drop immersion oil on the top of the condenser, then place the sample slide on the object stage, and the immersion oil will be filled between the two. The condenser must be used with a 100× oil lens equipped with an adjustable diaphragm. Another medium-magnification dry darkfield condenser can be used with a medium-magnification objective lens. This condenser has a central light stop, and the illumination light can only enter the condenser through the light transmission ring between the light stop and the edge of the condenser. For a fully equipped phase contrast microscope, the phase contrast condenser phase ring used with the Ph3 objective lens can be used with phase contrast objectives of 10× and below to form a low-magnification dark field effect.

Previous article:Yi Jie bunting slogan at gas station
Next article:Lishu county 1 ST middle school teaching building sign