Basic steps of biological production

1. Learn and master the making method of temporary loading.

2. on the basis of completing the basic experimental requirements, according to your own interests, Make more temporary packing

Second background knowledge (click to expand)

Third experimental content

Make temporary packing of onion epidermal cells

Four experimental supplies

Eyedropper, gauze, tweezers, absorbent paper, glass slide, cover glass, clean water, blade, dye solution (iodine solution)

Five experimental materials

Onion. Cover glass (animation)

2. Drop a drop of clear water (animation) in the center of the glass slide (Figure ZX-21)

3. Draw a square of about 1cm2 on the inner epidermis of onion with a blade (pay attention to safety when using the blade), and tear off the inner epidermis of onion with tweezers (animation) (Figure ZX-22)

4. Place the inner epidermis on. And spread it out (animation) (Figure ZX-23)

5. Cover the cover glass slowly from one side, without bubbles (animation) (Figure ZX-24)

6. Drop a drop of dye solution (animation) on one side of the cover glass

7. Then use absorbent paper to absorb excess dye solution from the other side, so that onion epidermal cells can be dyed evenly. (Figure zx-25)

Dyed onion cells (showing nucleus) (Figure)

Precautions for microscope use

1. When moving the microscope, hold the mirror arm with one hand and the mirror base with two upper arms close to the chest wall. Do not lift it obliquely with one hand and swing it back and forth to prevent the lens or other parts from falling.

2. When observing the specimen, the microscope should keep a certain distance (5cm) from the edge of the experimental platform, so as to prevent the microscope from falling to the ground. The tilt angle between the mirror column and the mirror arm shall not exceed 45 degrees, and it shall be restored immediately after use.

3. When using, you should strictly follow the steps, be familiar with the performance of each part of the microscope, and master the relationship between the rotation direction of the coarse and fine adjustment buttons and the lifting of the lens barrel. When turning the coarse adjustment button downward, your eyes must look at the object lens.

4. When observing the temporary specimen with liquid, cover it, and do not use inclined joints, so as not to pollute the lens and microscope with liquid.

5. The coarse and fine adjustment buttons should be used together, and the fine adjustment button should not be excessively rotated in one direction. When adjusting the focal length, watch the lens barrel descend from the side to avoid crushing the specimen and lens.

6. When observing a specimen with a monocular microscope, both eyes should be opened at the same time, the left eye should observe the object image, the right eye should be used for drawing, the left hand should adjust the focal length, and the right hand should move the specimen or draw.

7. It is forbidden to unscrew or replace the eyepiece, objective lens and condenser at will.

8. There is dirt on the optical parts of the microscope, which can be wiped with mirror paper or silk cloth. Never wipe it with fingers, rough paper or handkerchief to avoid damaging the mirror surface.

9. All corrosive and volatile chemical reagents and drugs, such as iodine, ethanol solution, acids, alkalis, etc., should not come into contact with the microscope. If they are inadvertently contaminated, they should be wiped clean immediately. Do not take off the eyepiece at will to prevent dust from falling into the lens barrel.

1. after observing the sample with an oil mirror, immediately wipe the oil mirror lens and the carrier with xylene to prevent other objective glasses from being stained with fragrant asphalt. Xylene is toxic. Wash your hands immediately after use.

11. After the experiment, take out the glass slide, wipe the lens with mirror paper and move it away, not facing the light hole. Wrap it in silk and put it back in the mirror box. Never expose the microscope to direct light.

Important points for attention in microphotography operation

1. The photographer's adjustment of microphotography device includes the adjustment of the pupil distance between two eyepieces and the correction of personal ametropia. The former refers to adjusting the distance between the two eyepieces according to the individual pupil spacing, pulling the eyepieces or twisting the pupil spacing adjustment screw. When correcting refraction, the diopter adjusting ring on the eyepiece barrel should be rotated, so that the "X" in the visual field center of the objective lens is changed from single line to double line to achieve complete clarity, and the left and right eyes should be adjusted separately. Every photographer should not omit this step.

2. Selection of different combinations of objective lens and photographic eyepiece: The photographic eyepiece does not have spatial resolution except magnification, and only the objective lens has spatial resolution. Under normal conditions, if the thickness of tissue section is less than 2 μm, the objective lens with higher magnification should be selected as far as possible. For example, if you want to enlarge the object 5 times, you should choose the combination of "2×" objective lens and "2.5×" eyepiece, which has higher definition than the combination of "1×" objective lens and "5×" eyepiece. However, the thickness of the section and the specificity of the observed specimen should also be considered. For example, when observing the nerve fibers or blood vessels meandering on the frozen section with a thickness of more than 3 μm, the focal depth of the lower power objective lens is longer, which is beneficial to continuously observe and analyze the images of nerves or blood vessels that do not walk on the same plane from different depths, levels or angles. In this case, the objective lens and eyepiece can be combined in different ways to shoot for many times, and finally the one with the best effect can be selected.

3. The adjustment and use of the condenser: inexperienced photographers often lack the adjustment of the height of the condenser, and they don't know whether the light source deviates from the center of the field of view. Many people only focus on the so-called "up focus" between the objective lens and the tissue slice, but not on the "down focus" between the condenser and the tissue slice, which of course can't obtain the best definition imaging negative. The steps of Kohler illumination method are as follows: ① Minimize the aperture of the field of view, so that the through hole of the aperture blade presents its octagonal image; ② Twist the left and right centering screws under the stage with both hands to make the diaphragm image coincide with the central circle of the field of view to correct the light path; ③ Adjust the height of the condenser to make the octagonal diaphragm image clear from blur, that is, "down focus"; ④ Enlarge the diaphragm to the periphery of 135 frames (referring to the conventional 135 negative frame, 24 mm×36 mm). Fine-tune the height of the condenser again to make the diaphragm image clearest. Every time the magnification is changed, the above adjustment steps should be repeated.

4. Improve photographic contrast: The contrast of tissue structure depends on the quality of tissue preparation technology. This is an important link to improve the quality of photomicrography. However, in general, in order to make up for the lack of contrast in slicing, the following remedial measures can often be adopted: ① According to the numerical aperture value on the objective lens, that is, the NA value, the aperture stop of the condenser is adjusted accordingly. Generally, the objective lens with good quality is marked with the magnification and the NA value. The larger the NA value, the higher the spatial resolution. ② If the image contrast is still insufficient due to poor tissue preparation after adjusting the NA value dial in this way, the NA value of the aperture stop of the objective lens can be appropriately reduced, for example, the 1x objective lens can be adjusted to .19. (3) If the image contrast is still not good after the above measures, the field stop can be shrunk from the frame of 135 (referring to the conventional 135 photo frame of 24 mm×36 mm) to the frame, and then the field stop image can be removed when the darkroom is enlarged. Of course, the last two measures mentioned above are just a slightly modified remedy.

5. Low-power photography is difficult: Low-power photography has its special advantages, for example, under the magnification of 1 (object) ×2.5 (objective), it can shoot the whole picture of one side of rat brain slice, which has a clear effect on the distribution of specific markers, but the low-power objective lens has low resolution and long focal depth, so it is difficult to focus accurately with fine-tuning screw. In order to avoid individual differences in vision, we should take three steps: under-focusing, over-focusing and positive focusing, and focusing should not be repeated.

6. Use of oil-immersed lens: Most of the 1× objective lenses are oil-immersed lenses, however, after use, the lenses are often damaged due to poor wiping. The alternative method is to drop ultra-pure water or double distilled water, and the observation effect is not much different from that of fragrant asphalt. Because the distance between the 1× objective lens and the slice is very close, it is easy to damage the lens. You must focus on the 4× objective lens first, then turn to the 1× objective lens, and gently turn the focusing fine-tuning screw to the focus. In order to avoid lens damage, the new 1× objective lens often has a spring device, which can make the lens slightly expand and contract to avoid its damage.

7. Other precautions: ① According to the convention, LBD (color temperature conversion) filter should be added to color film, and IF55 (green) filter should be added to black and white film. LBD filter can make the solar color roll get the best color temperature compensation, and IF55 filter can make the spectral sensitivity of black and white roll close to that of human eyes. Although some people think that it is not necessary to filter, because photography depends on the chemical sensitivity of the film and does not depend on the intuitive feeling of people's eyes on the field of vision, it is better to add filters. ② The selection of exposure time is also a problem that must be paid attention to. It is known that there are many different combinations between light intensity and exposure time, which are within the "safe" range of exposure, but the so-called "safe" range is not equal to the best condition, so the author thinks that it is necessary to limit the exposure time. The reason is that the chemical sensitivity of a film is limited to some extent. If the exposure time of 36 frames of a film is changed at will, the difference between 36 frames will be great, and it is inevitable that some frames will be developed under the same conditions. According to the author's experience, the exposure time is generally limited to .5 ~ 1 s, and the image effect of the negative film is better. (3) to focus on the structure in the center of the field of view, because the exposure time measured by the automatic exposure device is based on the central area of the field of view, and the farther away from the central area, the more inaccurate it is. Sometimes, in order to give consideration to the local solution of the structure, and the key structure is not in the center of the field of view, the spot exposure method can be adopted. (4) If the structure on a slice needs to be shot into several pieces and then spliced, a lock key can be set on the New Vanox microphotograph, which is beneficial to solve this problem, so as to make the exposure time of several frames of the same structure consistent, and then put this group of photos into developing solution and fixing solution at the same time when developing photos.