What are the sample requirements for light microscopy?
In the sample preparation process of the optical microscope, the thickness of the slice of the sample is between 2 and 25um; the thickness of the slice of the electron microscope is within 50-100nm (except for the high-voltage electron microscope, the thickness of the sample can reach 1um), so the slicing method used is not all the same.
In terms of the carrier, the carrier of the optical microscope section is a glass slide, and the carrier of the electron microscope section is the carrier grid.
In terms of fixation, optical microscope sections are fixed with a compound fixative, while electron microscope sections are repeatedly fixed with a single fixative
In terms of staining, the staining of optical samples is relatively simple. According to different observation samples and the type of optical microscope, it is usually enough to use some fixed stains. There are many methods for re-staining electron microscope samples, and they are also very complicated, 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 make the field of view of the microscope receive uniform and sufficient illumination, it is necessary to adjust the illumination optical path system when the microscope is first installed and debugged. requirements. In addition, correctly grasping the adjustment of the illumination optical path system is a necessary step after replacing the light bulb in the process of using the microscope, and it is also a necessary means to test the performance of the microscope from time to time in the daily use process. The adjustment of the microscope illumination optical path system mainly includes the following 4 items: 1. Preliminary adjustment of the lighting source lamp chamber outside the microscope ① First, open the shell of the lamp chamber, and put the halogen bulb into the socket by pressing the spring clip. Dirt such as fingerprints left on the bulb will affect the life of the bulb. ②Place the lamp room on the table, after turning on the power, use a special screwdriver to adjust the focusing 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 to adjust the filament hole (marked with "──") 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 position of the filament appropriate .
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 illuminator into the field of view of the objective lens, and to ensure that the field of view of the microscope is fully and uniformly received from the perspective of the light source. Lighting, which is a prerequisite for adjusting the Kühler lighting system. Basic tools required: Centering telescopes are equipped with microscope purchase. ① Unplug the frosted glass sleeve in the lamp house and put the lamp house back on the microscope ② Select a 10× objective lens, turn on the light source program to find the sample and adjust the focus clearly, and then use a 40× objective lens to focus the sample clearly (40× objective lens The whole picture of the filament can be seen 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 a centering telescope, grab the white part, and extend the black eyepiece with the other hand, The filament image can be seen in the field of vision; ⑤If the filament position is not suitable, adjust the "──" hole, adjust the filament image in the horizontal direction, adjust the "──" hole, and adjust the filament image in the vertical direction until Adjust the filament image to a circular image that just fills the aperture of the objective lens; ⑥ After the adjustment, insert the frosted glass sleeve back to its original position, unplug the centering telescope, and replace the eyepiece for the next adjustment. The above-mentioned adjustment of the illumination light source lamp room outside the microscope and the calibration of the position of the light source illuminator inside the microscope only need to be carried out when the microscope is installed and debugged for the first time and the bulb is replaced. In case of confusion, you can restore it to the original state according to the above steps.
3. Correct adjustment of the Kohler illumination (Kohler) system Correct adjustment of the microscope, one of the main tasks is the adjustment of the illumination light path system, and the key is the adjustment of the Kohler illumination system. For everyone who uses microscopes, especially those who take micrographs, they should have a certain understanding and mastery of the principles of the Kühler illumination system and its adjustment steps, so that they can give full play to the functions of the microscope. The resulting photos can be more consistent and perfect in effect. The principle of the Kühler illumination system is simply: the light emitted by any point on the light source illuminator can illuminate the field of view of the microscope, and the light emitted by each point on the light source illuminator is collected, and it is in the field of view of the microscope. A very sufficient and uniform illumination is achieved. The purpose of adjusting the Kuhler illumination 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 the formation of fog on the negative during photography. Essential components of a high-adjustment Kühler illumination system: field diaphragm, condenser system with on-axis adjustment. 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 not too 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 A locally illuminated area or bright spot will appear in the field of view, which is a blurred image of the field diaphragm, in which the details of the sample can be clearly seen; See the details of the sample clearly
⑥ Lower the condenser slightly, so that the bright spot in the field of view gradually narrows and gradually becomes a clear polygonal image, which is the clear image of the field diaphragm;
⑦ Under normal circumstances, the polygonal image is not in the center of the field of view. It is necessary to adjust a pair of centering screws of the condenser to adjust the polygonal image of the field of view to the center position;
⑧ Gradually open the field of view diaphragm to make the polygonal image become an inscribed polygon of the field of view, and further check the alignment condition. If the alignment is not ideal, continue to adjust the alignment screw slightly;
⑨ Slightly open the field diaphragm slightly to make the polygonal image disappear on the edge of the field of view. At this point, the adjustment of the Kühler lighting system is completed. After the Kühler illumination system was adjusted, the entire field of view was illuminated evenly, and the micrographs were bright and clear with normal contrast.
