What sample specifications are needed for light microscopy?
In the sample preparation process of the optical microscope, the slice thickness of the sample is between 2-25um; the slice thickness of the electron microscope is within 50-100nm (except for the high-voltage electron microscope, whose sample thickness can reach 1um), so the slice method used is not all the same.
In terms of carrier, the carrier of optical microscope section is glass slide, while the carrier of electron microscope section is grid.
In terms of fixation, the slices of the optical microscope are fixed with a compound fixative, while the slices of the electron microscope are fixed repeatedly with a single fixative.
In terms of staining, the staining of optical samples is relatively simple. According to different observation samples and types of optical microscopes, it is usually enough to stain with some fixed staining agents. There are many and complicated methods for re-staining electron microscope samples, such as negative staining, silver staining and so on.
In terms of embedding, paraffin, collodion, gelatin, etc. are used as embedding agents for optical microscope sections; epoxy resin, polystyrene resin, isobutylene resin, and water-soluble resin are used as embedding agents for electron microscope sections.
In order to ensure that the field of view of the microscope can be evenly and fully illuminated, the illumination optical system must be adjusted when the microscope is first installed and debugged. This is an important means and the most basic way to use the microscope correctly and obtain correct and reliable results. requirements. In addition, correctly mastering the adjustment of the illumination optical path system is a necessary step after replacing the light source bulb during the use of the microscope, and it is also a necessary means to check the performance of the microscope from time to time during daily use. The adjustment of the microscope illumination optical path system mainly includes the following four items: 1. Preliminary adjustment of the light source lamp chamber outside the microscope ① Open the shell of the lamp chamber first, press the spring clip to put the halogen bulb into the socket, and avoid direct contact with the bulb with your fingers during installation (can be separated by soft cloth or paper) to avoid There are fingerprints and other dirt on the bulb, which will affect the service life of the bulb. ②Put the lamp house on the table, after turning on the power, use a special screwdriver to adjust the focus knob hole of the lamp (marked with "←→"), so that the filament is projected on the wall 1-2m away, and the filament image is adjusted. Then adjust the height of the lamp and adjust the thread hole (marked with "──") to make the position of the filament appropriate; then adjust the left and right position of the lamp to adjust the screw hole (marked with "──") to make the left and right position of the filament appropriate .
2. The purpose of checking and correcting the position of the light source illuminant (filament) in the microscope is to adjust the image end of the illuminant squarely into the field of view of the objective lens, and to ensure that the field of view of the microscope is fully and uniformly illuminated from the perspective of the light source. Lighting, which is a prerequisite for adjusting the Kuhler lighting system. Basic tools needed: The centering telescope is equipped when the microscope is purchased. ① Unplug the frosted glass sleeve in the lamp house, and put the lamp house back on the microscope. ② Choose a 10× objective lens, turn on the light source program to find the sample and focus it clearly, and then use a 40× objective lens to focus the sample clearly (40× objective lens You can see the whole picture of the filament); ③ Open the aperture diaphragm and field diaphragm of the condenser to the maximum; ④ Unplug one of the eyepieces, replace with a centering telescope, grasp the white part, stretch the black eyepiece with the other hand, You can see the filament image in the field of vision; ⑤ If the position of the filament is not suitable, adjust the "──" hole to adjust the filament image in the horizontal direction, and adjust the "──" hole to adjust the filament image in the vertical direction until Adjust the filament image to the light circular image that just fills the aperture of the objective lens; ⑥ After the adjustment, insert the frosted glass sleeve back to the original position, unplug the centering telescope, and replace the eyepiece for the next adjustment. The above-mentioned adjustment of the illumination light source lamp chamber outside the microscope and the verification of the position of the light source luminous body inside the microscope only need to be carried out when the microscope is first installed and debugged and when the bulb is replaced, and the microscope cannot be adjusted randomly when using the microscope. In case of confusion, it can be adjusted back to the original state according to the above steps.
3. Correct adjustment of the Kohler illumination system One of the main tasks of the correct adjustment of the microscope is the adjustment of the illumination optical path system, and the key is the adjustment of the Kohler illumination system. For every person who uses a microscope, especially those who do photomicrographs, they should have a certain understanding and mastery of the principle of the Kuhler illumination system and its adjustment steps in order to give full play to the functions of the microscope and take pictures. The photos that come out can be more consistent and perfect in effect. The principle of the Kohler illumination system is simply: the light emitted by any point on the light source illuminant can illuminate the field of view of the microscope, and the light emitted by each point on the light source illuminant is collected, and in the field of view of the microscope A very full and uniform illumination is achieved. The purpose of adjusting the Kuhler lighting system is to obtain uniform and sufficient illumination for the observed field of view, and to prevent stray light from affecting or interfering with the imaging system, so as to avoid fogging on the film during photography. Necessary components for a high-adjustment Kohler illumination system: field diaphragm, condenser lens system that can be adjusted on-axis. ① Select 10× objective lens and 10× eyepiece ② Put the front lens of the condenser into the optical path, adjust the aperture diaphragm to a moderate position (not too big or small), then raise the condenser to the top position, and adjust the condenser turntable to Bright field "J" position ③ Adjust the field diaphragm to the minimum (0.1)
④ Put the sealed biological sample on the stage, turn on the light source, and focus clearly
⑤ There will be a partially illuminated area or bright spot in the field of view, which is the blurred image of the field diaphragm, in which the details of the sample can be clearly seen; outside it is a darker field of view, which may not necessarily See the details of the sample clearly
⑥Slightly adjust the condenser downwards, so that the bright spot in the field of view gradually shrinks, and gradually becomes a clear polygonal image, which is the clear image of the field diaphragm;
⑦Generally, the polygonal image is not in the center of the field of view, and it is necessary to adjust a pair of centering screws of the condenser to adjust the polygonal image of the field diaphragm to the central position;
⑧Gradually open the diaphragm of the field of view to make the polygon image an inscribed polygon of the field of view, and further check the alignment status. If the alignment is not ideal, continue to fine-tune the alignment screw;
⑨Slightly open the diaphragm of the field of view, so that its polygonal image just disappears on the edge of the field of view. So far, the adjustment of the Kohler illumination system is completed. After the Kohler lighting system is adjusted, the entire field of view is evenly illuminated, and the micrographs taken are bright and clear with normal contrast.
