Composition structure of optical microscope

Optical system, the optical system of microscope mainly includes objective lens, eyepiece, reflector and condenser. Broadly speaking, it also includes light source, filter, cover glass and glass slide. (a), the objective lens, the objective lens is a very important component to determine the performance of the microscope, installed on the objective lens converter, close to the observed object, so it is called the objective lens or objective lens. The magnification of the objective lens is proportional to its length. The greater the magnification of the objective lens, the longer the objective lens. 1. The classification of objective lens can be divided into dry objective lens and immersion objective lens according to different use conditions; Among them, immersion objective lens can be divided into immersion objective lens and oil-immersed objective lens (the common magnification is 90- 100 times). According to the different magnification, it can be divided into low-power objective (10 times or less), medium-power objective (about 20 times) and high-power objective (40-65 times). According to aberration correction, it can be divided into achromatic objective lens (commonly used, which can correct the chromatic aberration of two colors in the spectrum) and apochromatic objective lens (which can correct the chromatic aberration of three colors in the spectrum, which is expensive and rarely used). 2. Main parameters of objective lens: The main parameters of objective lens include magnification, numerical aperture and working distance. ① Magnification refers to the ratio of the size of the image seen by the eyes to the size of the corresponding specimen. It refers to the ratio of length, not the ratio of area. Example: The magnification is 100×, which refers to the specimen with the length of 1 μ m, and the length of the magnified image is100 μ m. If calculated by area, the magnification is 10000 times. The total magnification of the microscope is equal to the product of the magnification of the objective lens and the eyepiece. ② Numerical aperture, also called aperture ratio, abbreviated as NA or A, is the main parameter of objective lens and condenser, which is directly proportional to the resolution of microscope. The numerical aperture of dry objective lens is 0.05-0.95, and the numerical aperture of oil-immersed objective lens (fragrant asphalt) is 1.25. ③ Working distance refers to the distance from the lower part of the front lens of the objective lens to the upper part of the specimen cover glass when the specimen is clearly observed. The working distance of the objective lens is related to its focal length. The longer the focal length of the objective lens, the lower the magnification and the longer the working distance. Example: 10/0.25 and 160/0. 17 are marked on the objective lens, where 10 is the magnification of the objective lens; 0.25 is the numerical aperture; 160 is the lens barrel length (mm); 0. 17 is the standard thickness of cover glass (unit: mm). The effective working distance of 10x objective lens is 6.5mm, and that of 40x objective lens is 0.48mm. 3. The objective lens is used to enlarge the specimen for the first time. It is a very important part that determines the performance of microscope-resolution. Resolution is also called resolution or resolution. Resolution is expressed by the value of resolution distance (the minimum distance that can be resolved between two objects). At the apparent distance (25cm), the normal human eye can clearly see two objects with a distance of 0.073 mm, and this value of 0.073 mm is the resolution distance of the normal human eye. The smaller the resolution distance of the microscope, the higher the resolution, which means the better the performance. The resolution of microscope is determined by the resolution of objective lens, which is determined by its numerical aperture and the wavelength of illumination light. The resolution distance of the microscope is d=0.6 1λ/NA when using the ordinary central illumination method (bright illumination method that makes the light pass through the specimen evenly). Where d is the resolution distance of the objective lens in nm. λ —— wavelength of illumination light, in nanometer (nm). Numerical aperture of objective lens. For example, the numerical aperture of oil-immersed objective lens is 1.25, and the wavelength range of visible light is 400-700 nm. If the average wavelength is 550 nm, d=270 nm, which is about half the wavelength of illumination light. Generally, the resolution limit of a microscope illuminated by visible light is 0.2 micron. (2) The eyepiece is also called eyepiece because it is close to the observer's eyes. Mounted on the upper end of the lens barrel. 1. The structure of the eyepiece usually consists of an upper lens and a lower lens. The upper lens is called objective lens, and the lower lens is called convergent lens or field lens. There is an aperture between the upper and lower lenses or under the field lens (its size determines the field of view). Because the specimen is only imaged on the surface of the diaphragm, a short piece of hair can be stuck on the diaphragm as a pointer to indicate the target of a certain feature. Eyepiece micrometer can also be placed on it to measure the size of the observed specimen. The shorter the length of the eyepiece, the greater the magnification (because the magnification of the eyepiece is inversely proportional to the focal length of the eyepiece). 2. The function of the eyepiece is to further enlarge the clear real image that has been enlarged by the objective lens, so that the human eye can easily distinguish it clearly. The magnification of ordinary eyepiece is 5- 16 times. 3. The fine structure whose relationship between the eyepiece and the objective lens has been clearly distinguished by the objective lens cannot be seen clearly unless it is enlarged by the eyepiece. However, the fine structure that can't be resolved by the objective lens can't be seen clearly even if it is magnified by the high-power eyepiece, so the eyepiece can only play the role of magnification and will not improve the resolution of the microscope. Sometimes, although the objective lens can distinguish two close objects, it is still unclear because the image distance between the two objects is smaller than the resolution distance of the eyes. Therefore, the eyepiece and the objective lens are interrelated and mutually restricted. (3) Concentrator, also called concentrator. On the condenser bracket under the sample. It mainly consists of a condenser and an aperture. Among them, the condenser can be divided into bright field condenser (common microscope configuration) and dark field condenser. 1. The main parameter of optical lens, and the numerical aperture (NA) is the main parameter of condenser. The super-large numerical aperture is generally 1.2- 1.4, and the numerical aperture has a certain variable range. Usually, the number engraved on the upper lens holder represents a very large numerical aperture. By adjusting the opening of the lower iris, various numerical apertures below this value can be obtained. Some condenser lenses are composed of several groups of lenses, and the last group of lenses can be removed or moved out of the optical path, so that the numerical aperture of the condenser lens becomes smaller to adapt to the illumination when observed by a low-power objective lens. 2, the role of the condenser. The condenser is equivalent to a convex lens, which condenses light to enhance the illumination of the specimen. Generally speaking, the focal point of the condenser is designed to be about 65438 0.25mm above the lens plane at its upper end. (The focus is on the specimen to be observed, and the thickness of the slide is about 1. 1 mm). 3.iris, also known as aperture, is located under the condenser and consists of more than a dozen pieces of metal with a round hole in the middle. Its function is to adjust the light intensity, so that the numerical aperture of the condenser is adapted to the numerical aperture of the objective lens. The larger the aperture, the larger the numerical aperture (adjust the aperture to a large size after observation). Under the iris, there is also a circular filter bracket. Note: Only the teachers' microscope (1600× or 1500×) in the middle school laboratory is equipped with a condenser, and the students' microscope (640× or 500×) is equipped with a rotating diaphragm. Close to the stage, a circular disk that can rotate in a circle, a rotating diaphragm (also known as a shutter), and circular holes with different sizes on the diaphragm are called apertures. The diameters are 2, 3, 6, 12 and 16 mm respectively. Rotate the rotating diaphragm, and each aperture on the diaphragm can be aligned with the aperture, and the intensity of light can be adjusted through different apertures. (4) The reflector is a kind of double-sided mirror that can rotate at will, with a diameter of 50 mm, one side is flat and the other side is concave, and its function is to reflect the light coming from any direction through the light-passing hole. The flat mirror has weak reflective ability, so it is used when the light is strong, and concave mirror has strong reflective ability, so it is used when the light is weak. The reflector is usually a flat mirror on one side and a concave mirror on the other side, which is installed under the condenser and can rotate freely in both horizontal and vertical directions. The function of the reflector is to make the light or natural light emitted by the light source shine on the condenser. When using a condenser, generally use a flat mirror, and use a concave mirror when not in use; Use a flat mirror when the light is strong, and use a concave mirror when it is weak. After the observation, the reflector should be placed vertically. (5) Lighting source. Microscopes can be illuminated by natural or artificial light sources. 1. Natural light source, the light comes from the sky and is well reflected by white clouds. Do not use direct sunlight. 2. Artificial light source: ① Basic requirements of artificial light source: sufficient luminous intensity; The light source cannot generate too much heat. ② Common artificial light source: microscope lamp; The filt of that fluorescent lamp (6) is installed between the light source and the condense. The function is to let the light in the selected band pass and absorb other light, that is, to change the spectral composition of light or weaken the intensity of light. Divided into two categories: optical filters and liquid filters. (seven) cover glass and glass slide, cover glass and glass slide surface should be quite flat, no bubbles, no scratches. Just choose a colorless and transparent one, and wash it before use. The standard thickness of cover glass is 0.170.02 mm. If cover glass is not used or the thickness of cover glass is not appropriate, the imaging quality will be affected. The standard thickness of the chute is 1. 10.04 mm, and the general usable range is1-1.2 mm. Too thick will affect the efficiency of the concentrator, while too thin will easily break. Mechanical device, the mechanical device of microscope is an important part of microscope. Its function is to fix and adjust the optical lens, fix and move the specimen, etc. It mainly consists of a lens frame, a lens arm, an object stage, a lens barrel, an objective lens converter and a focusing device. (a), mirror base and mirror arm, 1. The mirror base is used to support the whole microscope. It is equipped with mirrors, and some are also equipped with illumination sources. 2. The function of the mirror arm is to support the lens barrel and the stage. There are two types: fixed and tiltable. ② Stage (also known as workbench and mirror stage). The stage is used for placing slides, and it has two shapes: round and square, in which the square has an area of120mm×110mm. There is a lamp hole in the center, and there are two small holes for installing flat clips on the left and right sides behind the lamp hole. There are two kinds: fixed and mobile. Some stages are equipped with cursors in vertical and horizontal coordinates, with a general reading of 0.1mm. The cursors can be used to determine the size of the sample or to mark the parts to be inspected. (3) a lens barrel, wherein the upper end of the lens barrel is provided with an eyepiece, and the lower end is connected with an objective lens converter. There are two types: fixed and adjustable. The length of the mechanical lens barrel (the distance from the upper edge of the eyepiece barrel to the lower end of the screw hole of the objective lens converter is called the lens barrel length or the mechanical lens barrel length) is called the fixed lens barrel, and the adjustable lens barrel can be replaced. Most new microscopes use fixed lens barrel, and most domestic microscopes also use fixed lens barrel. The mechanical lens barrel length of domestic microscopes is usually 160mm. There are two kinds of lens barrels for installing eyepieces: single barrel and double barrel. Single cylinder can be divided into vertical and inclined type, and double cylinder is inclined type. Among them, binocular microscope can observe both eyes at the same time, reducing eye fatigue. The distance between the two lens barrels can be adjusted, and one of the eyepieces has a diopter adjustment device, which is convenient for observers with different eyesight. (4) The objective lens converter is fixed at the lower end of the lens barrel and has 3-4 objective lens screw ports. The objective lenses should be arranged in the order of magnification. When rotating the objective lens converter, you should hold the rotating disk with your fingers and rotate it. Don't push the objective lens with your fingers, because pushing it for a long time will easily skew the optical axis and deteriorate the imaging quality. (5) Focusing device. The microscope is equipped with coarse focusing screws and fine focusing screws. Some microscopes have coarse quasi-focus spirals mounted on the same axis, with the large spiral being the coarse quasi-focus spiral and the small spiral being the fine quasi-focus spiral. Other separate arrangements. The larger pair of screws at the upper end of the lens arm are coarse focus screws, and the lens barrel rises or falls 10 mm every time it rotates. A pair of smaller screws located below the coarse focus screw are fine focus screws. When it rotates once, the lifting value of the lens barrel is 0.65438 0.8 mm, and the focusing range of the precision focus screw is not less than 65438 0.8 mm. Usage: 1 When using a monocular microscope, you should get into the habit of observing with your left eye (because you usually draw with your right hand). When observing, open your eyes at the same time, and don't close one eye, because it is easy to get tired. In order to train students to open their eyes at the same time, you can cut a rectangular piece of hard paper about 14cm long and 6cm wide, dig a round hole with a diameter slightly smaller than its outer diameter at the upper end of the lens barrel near the left end, put the round hole on the upper section of the lens barrel, open your eyes at the same time during observation, and block the line of sight of the right eye with the right end of the paper. After a period of training, we can get used to opening our eyes at the same time and then taking the paper away. 2. The joint between the mirror arm and the mirror base of the straight tube microscope is a mechanical joint, which can be used to adjust the inclination of the lens barrel for easy observation. The lens arm should not be tilted back too much, generally not more than 40. However, when using temporary loading observation (when the lens barrel tilts, the stage tilts, and the liquid on the slide is easy to flow out), it is forbidden to use tilting joints, especially when loading acidic reagents, so as not to pollute the lens body. 3. The use of eyepiece and objective lens, generally use the eyepiece with medium magnification (10×) and the objective lens with very low magnification to start observation, and gradually switch to the objective lens with higher magnification to find the magnification that meets the experimental requirements. When replacing the objective lens, first observe it with a low-power lens and adjust it to the correct working distance (clear imaging). If the high-power objective lens is further used for observation, before replacing the high-power objective lens, the part of the object image that needs to be enlarged for observation should be moved to the center of the field of view (when replacing the low-power objective lens with a high-power objective lens for observation, the object image range in the field of view is much reduced). The low power objective lens and the high power objective lens are basically in focus (focused at the same height). Generally speaking, when using any objective lens, the upper limit of effective magnification is 1 1,000 times its numerical aperture, and the lower limit is 250 times its numerical aperture. If the numerical aperture of the 40× objective lens is 0.65, the upper and lower limits are: 1000×0.65=650 times and 250×0.65≈ 163 times respectively. What exceeds the upper limit of effective magnification is called invalid magnification, which cannot improve the observation effect. The magnification below the lower limit cannot be distinguished by human eyes, which is not conducive to observation. Usually, the actual amplification range is 500-700 times the number between numerical apertures. 4. The use of oil-immersed objective lens, when using oil-immersed objective lens, generally do not use the same high focus. Focusing at the same height is only applicable to the original objective lens of each microscope, which is a very favorable and convenient condition when using low-power and high-power objective lenses, but it is limited when using oil-immersed objective lenses. Generally speaking, it is safe to focus on the same height when observing the specimen (slide) without cover glass with oil mirror, but it should be used with caution for the specimen with cover glass, because the working distance of the oil-immersed objective lens is very short. When the objective lens is immersed in oil, only aromatic tar drops on the specimen. After the observation, the cleaning work should be done in time. If it is not done in time, the fragrant tar will stick to dust, and dust particles may wear the lens when wiping. When the fragrant tar is exposed to the air for a long time, it will become thick and dry, which is very difficult to wipe, which is very unfavorable to the instrument. Wipe carefully. Wipe the front end of the oil-immersed objective lens once or twice with dry mirror paper to remove most of the oil stains, then wipe it twice with xylene dripping wet mirror paper, and then wipe it once with dry mirror paper. The fragrant asphalt on the specimen can be wiped off by "paper-pulling method" (that is, a small piece of mirror-wiping paper is covered on the fragrant asphalt, then some xylene is dropped on the paper, and the paper is pulled out while being wet, so that it can be wiped continuously for three or four times, and generally the smear specimen without cover glass will not be damaged). Mirror cleaning paper should also be dustproof. Generally, before use, each page is cut into 8 small pieces and stored in a clean Petri dish, which is economical and convenient to use. 5. How to use the condenser? ① The reason for using the condenser. When the magnification increases, on the one hand, the higher the magnification, the more lenses and the more light the lenses absorb. On the other hand, the brightness of the field of view (referring to the range where the specimen can be seen) is inversely proportional to the square of the magnification, that is, the higher the magnification, the darker the field of view. In order to get enough brightness, a condenser must be installed to focus the light on the specimen to be observed. (2) Observe the height of the condenser. When observing, in order to ensure good observation effect, the focus of the condenser should just fall on the specimen. In order to achieve this condition, it is necessary to adjust the height of the condenser. When irradiated with parallel light, the focus of the condenser is about 65438 0.25mm above the center of the upper lens plane. Therefore, when observing, it is often necessary to raise the condenser to a height where the upper plane of the lens is only slightly lower than the plane of the stage, so that the focus may fall on the specimen on the standard thickness slide. When using a glass slide thinner than the standard thickness to hold the specimen, the position of the condenser should be lowered accordingly, while when using a glass slide with too thick thickness, the focusing point can only fall below the specimen, which is not conducive to careful observation. ③ Coordination of condenser and objective lens. The so-called cooperation here is to make the numerical aperture of condenser and objective lens consistent, so as to make better and more detailed observation. If the numerical aperture of the condenser is lower than that of the objective lens, part of the numerical aperture of the objective lens is wasted, so that its high resolution cannot be achieved. If the numerical aperture of the condenser lens is larger than that of the objective lens, on the one hand, the specified resolution of the objective lens cannot be improved, on the other hand, the clarity of the object image will be reduced due to the wide illumination beam. The operation method of matching the condenser with the objective lens is as follows: after focusing, take off the eyepiece lens barrel, turn down the aperture under the condenser, and then slowly open it. Open the aperture just as large as the diameter of the field of view *, and then press the eyepiece to observe. Every time the objective lens is replaced, the matching operation should be carried out in turn. Some condenser aperture borders are engraved with scales indicating the aperture, which can be matched according to the scales. * Note: If the microscope uses an aperture stop instead of an aperture, the numerical aperture of the aperture stop (note that it is not the physical diameter of the lens) should be adjusted to 80% of the numerical aperture of the objective lens (see the center mark on the objective lens, for example, 10× the objective lens can be 0.30, etc.). ), a good balance can be achieved between large resolution and large contrast. The invention of microscope and every innovation of microscope in history have brought great progress to human cognition. It has brought unprecedented expansion to human life. Today, driven by scientific and technological innovation, the use of microscope has become a basic skill for middle school students. Mastering the structure, using it scientifically and maintaining it well will make it a window for teenagers to explore the future world. For more information, please consult Ningbo Zhan Jing Optical Instrument Co., Ltd., thank you!