Use and Maintenance of Biological Microscope
Biological microscopes are used to observe and study biological slices, biological cells, bacteria and living tissue culture, fluid precipitation, etc.
Can observe other transparent or translucent objects as well as powder, fine particles and other objects. Today let's talk about the use and post-production of biological microscopes
The use of a biological microscope
(1) Use environment and work habits
1. Use environment
The working place of the microscope should be clean, dry, free from vibration, and free from corrosive gases.
2. Work habits
(1) The height of the table and stool should be appropriate.
(2) During microscopic examination, even if a monocular microscope is used, both eyes must be opened at the same time, and the left eye must be observed. For drawing or recording with the right eye. If one eye is open and the other eye is closed, the eyes are prone to fatigue and cannot be watched for a long time. When working for a long time, you can take turns observing with two eyes.
(2) Preparation before use
1. Installation of optical system
For newly purchased microscopes or those whose optical system has been removed, the microscope must be installed before use. When installing, in order to prevent dust from falling downward, it should be installed in the order of first up and then down, that is, according to the order of eyepiece, objective lens, condenser lens and reflector. Figure 10-3-1 is the installation diagram of the microscope. Arrows show where the optics are mounted.
When installing the objective lens, the lens barrel should be raised first or the carrier stage should be lowered to keep a certain distance between the converter and the stage. Then, hold the objective lens, put it into the screw port of the converter, and rotate it slightly counterclockwise. After the objective lens is equipped with silk patterns, then screw it in clockwise until it is moderately tight. When installing the objective lens, it should be installed clockwise from small to large according to the magnification of the objective lens. When converting the objective lens, do not push the objective lens to rotate by hand, otherwise the optical axis of the objective lens will be skewed. It is best to hold the rotating disk of the converter by hand to rotate, or hold the knurled outer circle at the junction with the objective lens converter by hand to rotate.
After the eyepiece and objective lens are installed, insert the condenser into the condenser bracket under the stage. The height of the insertion should be such that when the condenser is raised to the highest point, the end face of the lens on the condenser is slightly lower than the plane of the stage, so as to prevent the glass slide from colliding with the lens of the condenser. Then, tighten the fixing screw of the condenser. For microscopes with non-electric light sources, the reflector is finally inserted into the socket under the condenser.
2. Calibrate the optical axis
The significance of correcting the optical axis is to make the main optical axis of the objective lens, eyepiece, condenser and the center point of the variable diaphragm coincide on a straight line. Therefore, it is also called coaxial adjustment or central adjustment. If the optical axis is skewed, the aberration will increase, and the resolution and sharpness will decrease.
The inspection method is to open the variable diaphragm to the maximum, screw the low-magnification objective lens into the optical axis, and lower the lens barrel so that the distance between the objective lens and the stage is smaller than the working distance of the objective lens (below 5mm). Without placing the specimen, adjust the angle of the reflector to make the field of view the brightest; or adjust the brightness of the light source lamp to make the field of view suitable for brightness and darkness.
Then, unplug the eyepiece and look directly through the tube. While slowly shrinking or opening the variable diaphragm several times, when the diaphragm is closed to the minimum, the image of the diaphragm (only a little bit at this time) should just fall on the center of the aperture of the objective lens. When the aperture is opened to a certain extent, the image of the aperture should coincide with the black circle of the aperture of the objective lens. If the above two conditions are met, it means that they are "coaxial". Otherwise, adjustments are required.
Both the eyepiece and objective are fixed and cannot be adjusted. Coaxial adjustment is mainly to adjust the position of the condenser. There are two optical axis correction screws on both sides of the condenser bracket of some microscopes, and the axis can be aligned by adjusting these two screws. Another kind of concentrator is supported by three screws 120° apart on the frame, one of which is equipped with a spring and can be stretched; the other two screws can be rotated. Adjust the two screws to fit the shaft.
After the optical axis of the microscope is calibrated, if the condenser is not removed or other special reasons, it is not necessary to calibrate frequently.
3. Prepare the specimen
Prepare high-quality specimens for later use.
(3) How to use reflectors and condensers and how to align light 1. Dimming
For microscopes using mirrors, flat mirrors are generally used to reflect the sun's scattered light. Concave reflectors should only be used in low light or when there are distractions outside the window.
For microscopes using electric light sources, just adjust the brightness appropriately.
2. Usage of concentrator
(1) Adjustment of the height of the condenser. In the case of parallel light irradiation, the focus of the general condenser is about 1.25mm above the center of the lens plane at its upper end. When using a high-magnification lens or an oil lens, due to the large magnification, the brightness of the mirror image is small, and strong lighting is required. Therefore, the condenser should be raised to the highest point so that the focus of the condenser just falls on the specimen plane. But when using a low magnification objective lens, the condenser can be properly lowered.
(2) Usage of the variable aperture The variable aperture plays two roles. One is to control the luminous flux directed at the specimen; the other is to change the numerical aperture of the condenser. Of the two roles, the latter is dominant. In order to make full use of the resolution of the objective lens, in principle, the numerical aperture of the condenser should be the same as that of the objective lens. Otherwise, resolution or clarity will be affected.
3. Lighting method
For the electric light source microscope, when using it, just adjust the light to the appropriate brightness. No aiming is required. However, for low-end microscopes using natural light, in order to obtain good observation results, the illumination light must be fully utilized. Therefore, the light should be adjusted before the microscope inspection. When facing the light, turn the low-magnification lens into the optical axis, raise the condenser appropriately, and open the variable diaphragm to the maximum. Then, look through the eyepiece while turning the mirror until the field of view is brightest and clearest. If you use natural light, try to avoid the interference of window frames and tree branches outside the window.
(4) Correct focusing of the objective lens
After the light is completed or the appropriate light is adjusted, raise the lens barrel or lower the stage, clamp the specimen slide on the mover, that is, the specimen holder, and move the part to be inspected to the center of the light hole of the stage. Then start focusing.
No matter what kind of inspection is done, it should start with a low power lens. When focusing, first use the coarse handwheel to lower the lens barrel so that the distance between the front lens of the low-magnification mirror and the cover glass is slightly smaller than the working distance of the objective lens (below 5mm). In order to avoid the objective lens pressing on the specimen slide, peep from the side. Then, while observing the field of view from the eyepiece, use the coarse handwheel to slowly raise the lens barrel. After seeing the object image for the first time, use the fine handwheel to fine-tune the focus until the object image is the clearest. The field of view of the low magnification objective lens is large, which is conducive to observing the whole picture of the specimen. You can also use the mover or adjust the handwheel vertically and horizontally to find the observed target. If necessary, the found target can be moved to the center of the field of view, ready for high-power lens observation.
When converting from a low-magnification objective lens to a high-magnification objective lens, if the objective lens is the original equipment of the microscope, and the slide glass and cover glass used meet the standards, "equal height conversion" can generally be performed. That is, after the conversion, you can see a clear image as long as you slightly adjust the fine-tuning knob. But the oil lens does not insist on parfocality. It is best to raise the lens barrel before switching, and finally refocus according to the focusing method of the low-power lens.
The method of using the oil lens is as follows: first raise the lens barrel, remove the specimen slide, lower the condenser slightly, and drop two drops of cedar oil on the lens of the condenser (there should be no air bubbles in the oil. If any, It can be removed with a small wooden stick), then put the specimen slide back to its original position, and raise the condenser so that the bottom surface of the slide is in contact with cedar oil. In this way, the oil immersion of the condenser is completed. Next, 1 drop of cedar oil was dropped on the coverslip. Then peep from the side, and use coarse adjustment to lower the lens barrel as far as possible until the front lens of the oil lens is immersed in cedar oil (but not yet in contact with the glass slide), thus completing the oil immersion of the objective lens. Then, while observing from the eyepiece, use the micro-handwheel to slowly raise the lens barrel (be careful not to twist the wrong direction and crush the cover glass) until the clearest object image appears in the field of view.
The oil immersion of the condenser can also adopt another method of dripping oil: that is, instead of directly dropping the oil on the lens of the condenser, turn the slide over and drop the oil on the bottom surface of the slide, and then Turn it over again, align and place it on the concentrator, and then raise the concentrator to complete the oil immersion of the concentrator. Although this method is not so smooth, it is safer. Some people use a glass rod to directly contact the condenser to apply cedar oil. This method is easy to scratch the lens and should not be used.
When using an oil lens, it is allowed not to add cedar oil between the condenser and the specimen, that is, air is still used as the medium on the condenser, but this will sacrifice the resolution of the objective lens.
If you need to switch back to the high-magnification objective lens for observation after using the oil lens, wipe off the oil on the cover glass to avoid contamination of the high-power objective lens. However, the oil on the concentrator does not need to be wiped, as long as the aperture is properly reduced a little.
After the oil lens is used, the cedar oil should be wiped clean in time. The lens can be wiped 1 or 2 times with a clean lens cleaning paper to wipe off most of the oil. Then wipe it twice with a lens tissue wetted with xylene, and finally wipe it clean with a lens tissue. The cleaning method of the condenser is the same. If the specimen needs to be preserved, the cedar oil on the glass slide can be wiped clean by "pulling paper". Cover the glass slide with lens cleaning paper, drop a drop of xylene on the paper, drag the paper strip while it is wet, and wipe it clean several times in a row.
Finally, point out: During the entire focusing process (especially the focusing of high magnification objective lens and oil lens), every movement should be carried out slowly. Otherwise, the object image will flash by and the target of observation cannot be found.
