Why are microscope images upside down?

As early as the first century BC, people have discovered that when observing small objects through spherical transparent objects, they can be magnified and imaged. Later, we gradually came to understand the law that the surface of spherical glass can make objects magnified and imaged. By 1590, Dutch and Italian eyeglass makers had created magnifying instruments similar to microscopes. Around 1610, while studying telescopes, Galileo of Italy and Kepler of Germany changed the distance between the objective lens and the eyepiece to arrive at a reasonable optical path structure for the microscope. The optical craftsmen at that time were engaged in the manufacture, promotion and improvement of microscopes. . In the mid-17th century, England's Hooke and Holland's Leeuwenhoek both made outstanding contributions to the development of microscopes. Around 1665, Hooke added coarse and fine focusing mechanisms, an illumination system, and a worktable for carrying specimens to the microscope. These components have been continuously improved and become the basic building blocks of modern microscopes. Between 1673 and 1677, Leeuwenhoek built single-component magnifying glass-type high-power microscopes, nine of which have survived to this day. Hooke and Leeuwenhoek used homemade microscopes to make outstanding achievements in the study of the microstructure of animals and plants. In the 19th century, the emergence of high-quality achromatic immersion objectives greatly improved the ability of microscopes to observe fine structures. Amici was the first to use a liquid immersion objective in 1827. In the 1870s, the German Abbe laid the classical theoretical foundation for microscope imaging. These promoted the rapid development of microscope manufacturing and microscopic observation technology, and provided powerful tools for biologists and medical scientists including Koch and Pasteur to discover bacteria and microorganisms in the second half of the 19th century. While the structure of the microscope itself is developing, microscopic observation technology is also constantly innovating: polarized light microscopy appeared in 1850; interference microscopy appeared in 1893; and in 1935, Dutch physicist Zernike created phase contrast microscopy. technique, for which he won the Nobel Prize in Physics in 1953. The classical optical microscope is just a combination of optical components and precision mechanical components. It uses the human eye as a receiver to observe the magnified image. Later, a photographic device was added to the microscope, using photosensitive film as a receiver that could be recorded and stored. In modern times, optoelectronic components, television camera tubes, and charge couplers are commonly used as receivers of microscopes, and coupled with microelectronic computers, they form a complete image information collection and processing system. At present, the world's most important microscope manufacturers include: Olympus, Zeiss, Leica, and Nikon. The main domestic manufacturers include: Jiangnan, McAudi, etc. 2. Basic optical principles of microscopes (1) Refraction and refractive index Light propagates in straight lines between two points in a uniform isotropic medium. When passing through transparent objects of different density media, refraction occurs. This is due to The propagation speed of light in different media is different. When light rays that are not perpendicular to the surface of a transparent object are incident on a transparent object (such as glass) from the air, the light ray changes direction at its interface and forms a refraction angle with the normal. (2) Performance of lenses Lenses are the most basic optical elements that make up the optical system of a microscope. Components such as objective lenses, eyepieces, and condensers are composed of single or multiple lenses. According to their different shapes, they can be divided into two categories: convex lenses (positive lenses) and concave lenses (negative lenses). When a beam of light rays parallel to the optical axis passes through a convex lens and intersects at a point, this point is called the "focus", and the plane that passes through the intersection point and is perpendicular to the optical axis is called the "focal plane". There are two focal points. The focus in object-space is called "object-space focus" and the focal plane there is called "object-space focal plane". On the contrary, the focus in image-space is called "image-space focus". The focal plane at is called the "image square focal plane". After light passes through a concave lens, it forms an upright virtual image, while a convex lens forms an upright real image. Real images can appear on the screen, but virtual images cannot. (3) Five imaging rules of convex lenses 1. When the object is located beyond twice the focal length on the object side of the lens, a reduced inverted real image is formed within twice the focal length on the image side and outside the focus; 2. When the object is located on the object side of the lens When the object is located within twice the focal length of the object side of the lens but outside the focus, an enlarged image will be formed beyond the double focal length of the image side. Inverted real image; 4. When the object is located at the object side focus of the lens, the image side cannot form an image; 5. When the object is located within the object side focus of the lens, there is no image formation on the image side, and on the object side of the lens A magnified upright virtual image is formed on the same side farther than the object. 3. Principle of imaging (geometric imaging) of optical microscopes Only when the opening angle of an object to the human eye is not less than a certain value, the naked eye can distinguish its details. This quantity is called visual resolution ε. Under optimal conditions, that is, when the illumination of the object is 50~70lx and the contrast is large, it can reach 1'. For easier observation, this amount is generally increased to 2', and this is taken as the average eyepiece resolution. The size of the object's visual angle is related to the length of the object and the distance from the object to the eye. There is a formula y=Lε, and the distance L cannot be made very small, because the adjustment ability of the eyes has a certain limit. Especially when the eyes work close to the limit range of the adjustment ability, the vision will be extremely fatigued. For standard (face view), the optimal viewing distance is 250mm (clear vision distance). This means that without instruments, eyes with a visual resolution of ε=2' can clearly distinguish the details of objects with a size of 0.15mm. When observing objects with a viewing angle less than 1', a magnifying instrument must be used.

Magnifying glasses and microscopes are used to observe objects that are placed close to the observer and should be magnified. (1) The imaging principle of a magnifying glass. An optical lens made of glass or other transparent materials with a curved surface can magnify objects and image them. The optical path diagram is shown in Figure 1. The object AB located within the object-side focus F has a size of y. It is magnified into a virtual image A'B' of a size of y'. The magnification rate of the magnifying glass is Γ=250/f', where 250--clear vision distance, in mm f'--focal length of the magnifying glass, in mm. The magnification rate refers to the image of an object observed with a magnifying glass within a distance of 250 mm. The ratio of the angle of view to the angle of view of an object observed without a magnifying glass. (2) The imaging principle of a microscope A microscope and a magnifying glass play the same role, which is to turn small nearby objects into a magnified image for observation by the human eye. It's just that a microscope can have higher magnification than a magnifying glass. Figure 2 is a schematic diagram of an object being imaged by a microscope. For convenience, the figure shows the objective lens L1 and the eyepiece L2 as a single lens. Object AB is located in front of the objective lens, and its distance from the objective lens is greater than the focal length of the objective lens, but less than twice the focal length of the objective lens. Therefore, after it passes through the objective lens, it will inevitably form an inverted magnified real image A'B'. A'B' is located at the object focus F2 of the eyepiece, or very close to F2. It is then magnified into a virtual image A''B'' through the eyepiece for observation by the eyes. The position of the virtual image A''B'' depends on the distance between F2 and A'B', which can be at infinity (when A'B' is located on F2) or at the observer's apparent distance ( When A'B' is to the right of focus F2 in the figure). Eyepieces function like a magnifying glass. The only difference is that what the eye sees through the eyepiece is not the object itself, but the image of the object that has been magnified once by the objective lens. (3) Important optical technical parameters of the microscope During microscope inspection, people always hope to have a clear and bright ideal image, which requires the various optical technical parameters of the microscope to reach certain standards, and it is required that they must be used according to the The purpose of microscopy and the actual situation are used to coordinate the relationship between various parameters. Only in this way can we give full play to the performance of the microscope and obtain satisfactory microscopic examination results. The optical technical parameters of the microscope include: numerical aperture, resolution, magnification, focal depth, field of view width, coverage difference, working distance, etc. These parameters are not always higher, the better. They are interconnected and restrictive. When used, the relationship between parameters should be coordinated according to the purpose of microscopy and the actual situation, but the resolution should be guaranteed. . 1． Numerical Aperture Numerical aperture is abbreviated as NA. Numerical aperture is the main technical parameter of the objective lens and condenser lens. It is an important symbol for judging the performance of both (especially the objective lens). The numerical values ??are marked on the housings of the objective lens and condenser respectively. Numerical aperture (NA) is the product of the refractive index (n) of the medium between the objective front lens and the object being inspected and the sine of half the aperture angle (u). The formula is expressed as follows: NA=nsinu/2 The aperture angle, also known as the "lens angle", is the angle formed by the object point on the optical axis of the objective lens and the effective diameter of the front lens of the objective lens. The larger the aperture angle, the greater the light entering the objective lens. It is directly proportional to the effective diameter of the objective lens and inversely proportional to the distance from the focus. When observing under a microscope, if you want to increase the NA value, the aperture angle cannot be increased. The only way is to increase the refractive index n value of the medium. Based on this principle, water immersion objective lenses and oil immersion objective lenses are produced. Since the refractive index n value of the medium is greater than 1, the NA value can be greater than 1. The maximum value of numerical aperture is 1.4, which reaches the theoretical and technical limit. At present, bromonaphthalene with high refractive index is used as the medium. The refractive index of bromonaphthalene is 1.66, so the NA value can be greater than 1.4. It must be pointed out here that in order to give full play to the numerical aperture of the objective lens, the NA value of the condenser should be equal to or slightly larger than the NA value of the objective lens when observing. Numerical aperture is closely related to other technical parameters. It determines and affects almost all other technical parameters. It is directly proportional to the resolution, directly proportional to the magnification, and inversely proportional to the focal depth. As the NA value increases, the field of view width and working distance will decrease accordingly. 2. Resolution The resolution of a microscope refers to the minimum distance between two object points that can be clearly distinguished by the microscope, also known as the "discrimination rate". The calculation formula is σ=λ/NA, where σ is the minimum resolution distance; λ is the wavelength of light; NA is the numerical aperture of the objective lens. It can be seen that the resolution of the objective lens is determined by two factors: the NA value of the objective lens and the wavelength of the illumination source. The larger the NA value, the shorter the wavelength of the illumination light, the smaller the σ value, and the higher the resolution. To improve the resolution, that is, reduce the σ value, the following measures can be taken (1) Reduce the wavelength λ value and use a short-wavelength light source. (2) Increase the medium n value to increase the NA value (NA=nsinu/2). (3) Increase the aperture angle u value to increase the NA value. (4) Increase the contrast between light and dark. 3. Magnification and effective magnification: Due to the two magnifications of the objective lens and the eyepiece, the total magnification Γ of the microscope should be the product of the objective lens magnification β and the eyepiece magnification Γ1: Γ=βΓ1 Obviously, compared with the magnifying glass, the microscope can have Much higher magnification, and the magnification of the microscope can be easily changed by exchanging objective lenses and eyepieces with different magnifications. Magnification is also an important parameter of a microscope, but you cannot blindly believe that the higher the magnification, the better. The limit of microscope magnification is the effective magnification.

Resolution and magnification are two different but related concepts. The relevant formula is: 500NA<Γ<1000NA. When the numerical aperture of the selected objective lens is not large enough, that is, the resolution is not high enough, the microscope cannot distinguish the fine structure of the object. At this time, even if the magnification is increased excessively, only one can be obtained. Images with large outlines but unclear details are called invalid magnification. On the other hand, if the resolution meets the requirements but the magnification is insufficient, then although the microscope has the ability to resolve, the image is still too small to be clearly seen by the human eye. Therefore, in order to give full play to the resolving power of the microscope, the numerical aperture should be reasonably matched to the total magnification of the microscope. 4. Depth of focus Depth of focus is the abbreviation of depth of focus, that is, when using a microscope, when the focus is on an object, not only can all points on the plane at that point be seen clearly, but also within a certain thickness above and below this plane, If you can see clearly, the thickness of this clear part is the depth of focus. If the focal depth is large, the entire layer of the object being inspected can be seen, while if the depth of focus is small, only a thin layer of the object being inspected can be seen. The depth of focus has the following relationship with other technical parameters: (1) Depth of focus and total magnification The magnification is inversely proportional to the numerical aperture of the objective lens. (2) The depth of focus is large and the resolution is reduced. Because the depth of field of a low-magnification objective lens is larger, it causes difficulties when taking photos with a low-magnification objective lens. Details will be covered in photomicrography. 5. Field Of View When observing a microscope, the bright circular range seen is called the field of view, and its size is determined by the field diaphragm in the eyepiece. The diameter of the field of view, also called the width of the field of view, refers to the actual range of the object being inspected that can be accommodated in the circular field of view seen under the microscope. The larger the field of view diameter, the easier it is to observe. There is the formula F=FN/β, where F: field diameter, FN: field number (Field Number, abbreviated as FN, marked on the outside of the eyepiece barrel), β: objective lens magnification. It can be seen from the formula: (1) The diameter of the field of view is proportional to the number of fields of view. (2) As the magnification of the objective lens is increased, the diameter of the field of view decreases. Therefore, if you can see the entire object under inspection with a low-magnification lens, you can only see a small part of the object under inspection if you switch to a high-magnification objective lens. 6. Poor Coverage The optical system of a microscope also includes the coverslip. Due to the non-standard thickness of the cover glass, the optical path of the light refracted from the cover glass into the air changes, resulting in a phase difference, which is the coverage difference. The occurrence of poor coverage affects the sound quality of the microscope. According to international regulations, the standard thickness of a cover glass is 0.17mm, and the permitted range is 0.16-0.18mm. The difference in this thickness range has been taken into account in the manufacture of the objective lens. The mark 0.17 on the objective lens housing indicates the thickness of the cover glass required for the objective lens. 7. Working distance WD The working distance is also called the object distance, which refers to the distance between the surface of the front lens of the objective lens and the object being inspected. During microscopic examination, the object to be inspected should be between one and two times the focal length of the objective lens. Therefore, it and focal length are two different concepts. What we usually call focusing is actually adjusting the working distance. When the numerical aperture of the objective lens is constant, the shorter the working distance, the larger the aperture angle. A high-power objective lens with a large numerical aperture has a small working distance. (4) Objective lens The objective lens is the most important optical component of the microscope. It uses light to image the object under inspection for the first time. Therefore, it is directly related to and affects the quality of the image and various optical technical parameters. It is the primary criterion for measuring the quality of a microscope. . The objective lens has a complex structure and is precisely made. Due to the correction of object aberration, the metal objective lens barrel is composed of lens groups that are fixed at a certain distance apart. Objective lenses have many specific requirements, such as on-axis and parfocal. Parfocal means that during microscopic examination, when the image observed with an objective lens of a certain magnification is clear, when switching to an objective lens of another magnification, the image should also be basically clear, and the center deviation of the image should also be within a certain range. , that is, the degree of alignment. The quality of parfocal performance and the degree of alignment are an important symbol of microscope quality, which are related to the quality of the objective lens itself and the accuracy of the objective lens converter. Modern microscope objectives have reached a high degree of perfection, their numerical aperture is close to the limit, and the difference between the resolution in the center of the field of view and the theoretical value is negligible. However, the possibility of continuing to increase the field of view of the microscope objective and improve the imaging quality at the edge of the field of view still exists, and this research work is still ongoing today. There is a difference between microscope objectives and eyepieces in their participation in imaging. The objective lens is the most complex and important part of the microscope. It works in a wide beam (large aperture), but the inclination angle of these beams to the optical axis is small (small field of view); the eyepiece works in a narrow beam, but its inclination angle is large (field of view). The field is large). When calculating objective lenses versus eyepieces, there is a big difference in eliminating aberrations. The aberrations related to the wide beam are spherical aberration, coma aberration and positional chromatic aberration; the aberrations related to the field of view are astigmatism, field curvature, distortion and magnification package aberration. The microscope objective is an aspherical aberration system. This means that for a pair of yoke points on the axis, when spherical aberration is eliminated and sinusoidal conditions are achieved, there are only two such aspherical points per objective. Therefore, any change in the calculated positions of objects and images results in larger aberrations. 1． The main parameters of the objective lens (1) Magnification β (2) Numerical aperture NA (3) Mechanical tube length L: In a microscope, the distance between the objective lens supporting surface and the eyepiece supporting surface is called the mechanical tube length. For a microscope, the mechanical barrel length is fixed. Our country stipulates that the mechanical barrel length is 160 mm.

(4) Cover glass thickness d (5) Working distance WD These parameters are mostly engraved on the objective lens barrel, as shown in Figure 3. There is a so-called microscopic objective lens with an infinite tube length. This objective lens usually has an auxiliary objective lens (also called a compensation objective lens or a tube objective lens) behind the objective lens. The object being observed is located at the front focus of the objective lens. After passing through the objective lens, it is imaged at infinity. The image is then imaged on the focal plane of the auxiliary objective lens through the auxiliary objective lens, as shown in Figure 4. There is parallel light between the objective lens and the auxiliary objective lens, so the distance between them is relatively free, and optical elements such as prisms can be added. 2. Basic types of objective lenses (1) According to the length of the microscope barrel (in mm): 160 barrel for transmitted light, with a 0.17mm thick or thicker cover glass; 190 barrel for reflected light, without a cover glass; A lens tube is used for transmitted light and reflected light, and the tube length is infinite. (2) According to the characteristics of the immersion method: non-immersion type (dry type), immersion type (oil immersion, water immersion, glycerin immersion and other immersion methods). (3) According to optical device: transmission type, reflection type and catadioptric type. (4) According to numerical aperture and magnification: low magnification (NA≤0.2 and β≤10X), medium magnification (NA≤0.65 and β≤40X), high magnification (NA>0.65 and β>40X). (5) According to different aberration correction conditions, they are usually divided into achromatic objectives, semi-apochromatic objectives, apochromatic objectives, plano-achromatic objectives, plano-apochromatic objectives and monochromatic objectives. a. Achromatic objective: This is the most widely used type of microscope objective. It often has the word "Ach" on the outer shell. It corrects the positional chromatic aberration (red and blue), spherical aberration (yellow-green light) and sinusoidal difference of the point on the axis, maintaining the uniform brightness condition. The astigmatism at off-axis points does not exceed the allowable value (-4 is photometric), and the secondary spectrum is not corrected. Low-power achromatic objectives with numerical apertures of 0.1 to 0.15 are generally composed of a double-gel objective lens with two lenses glued together. Achromatic objectives with numerical apertures up to 0.2 are composed of two sets of doublet lenses. When the numerical aperture increases to 0.3, a plano-convex lens is added. This plano-convex lens determines the focal length of the objective lens, while other lenses compensate for the aberration caused by its plane and spherical surfaces. The plane aberration of high-magnification objective lenses can be eliminated by immersion method. High-power achromatic objectives are generally immersion-type and consist of four parts: a front lens, a crescent lens and two double plastic lens groups. b. Apochromatic objective has a complex structure. The lens is made of special glass or fluorite and other materials. The outer shell of the objective lens is marked with "Apo". It implements sinusoidal conditions for two colored lights, requiring strict correction of the positional chromatic aberration (red and blue dichromatic), spherical aberration (red and blue dichromatic) and sinusoidal difference of the point on the axis, and also requires correction of the secondary spectrum (re-correction Positional chromatic aberration of green light). The chromatic aberration of magnification cannot be completely corrected and must generally be compensated with an eyepiece. Due to the extremely complete correction of various aberrations, it has a larger numerical aperture than the achromatic objective lens with response magnification, which not only has high resolution and excellent image quality, but also has a higher effective magnification. Therefore, apochromatic objectives are high performance and suitable for advanced research microscopy and photomicrography. c. Semi apochromatic objective (Semi apochromatic objective) Semi-apochromatic objective is also called fluorite objective. The outer shell of the objective lens is marked with "FL". In terms of structure, the number of lenses is more than that of achromatic objective lens and less than that of apochromatic objective lens. In terms of imaging quality, it is far better than achromatic objective lens and close to that of apochromatic objective lens. d. Plan objective: Plan objective is to add a thick half-moon-shaped lens to the lens system of the objective lens to correct the defects of field curvature and improve the imaging quality at the edge of the field of view. Plan objectives have a flat field of view and are more suitable for microscopic examination and photomicrography. For planar field achromatic objectives, the chromatic aberration of magnification is not large and there is no need to use special eyepieces to compensate. For planar apochromatic objectives, eyepieces must be used to compensate for the chromatic aberration of magnification. e. Monochromatic objective lens This type of objective lens consists of a set of single lenses made of quartz, fluorite or lithium fluoride. It can only be used in certain areas of the ultraviolet spectrum (width not exceeding 20mm). Monochromatic objectives cannot be used in the visible spectrum. These objectives are made into reflective and catadioptric systems. The main disadvantage is that a considerable portion of the beam is blocked in the center (25% of the entrance pupil area). In the new catadioptric system, due to the use of a semi-transparent mirror and the glued structure of the objective lens, this shortcoming is greatly alleviated, so that the shading of the mirror frame can be eliminated. Moreover, the residual aberrations of the two coaxial mirrors are compensated for each other, and a lens group is used to increase the numerical aperture. If the calibration of the system is satisfactory and the aperture reaches NA=1.4, the central shielding may not exceed 4% of the entrance pupil area. f. Special objective lenses The so-called "special objective lenses" are specially designed and manufactured on the basis of the above-mentioned objective lenses to achieve certain specific observation effects. There are mainly the following types: (a) Correction collar objective is equipped with an adjustment ring in the middle of the objective lens. When the adjustment ring is rotated, the distance between the lens groups in the objective lens can be adjusted, thereby correcting the Poor coverage caused by non-standard coverslip thickness. The scale on the adjustment ring can range from 0.11--.023, and this number is also marked on the outer shell of the objective lens, indicating that the error in the thickness of the cover glass can be corrected from 0.11-0.23mm.

(b) An objective lens with an iris diaphragm (Iris diaphragm objective) is equipped with an iris diaphragm in the upper part of the objective lens barrel, and an adjustment ring that can also be rotated on the outside. When rotating, the size of the aperture of the diaphragm can be adjusted. This structure The objective lens is an advanced oil immersion objective lens. Its function is that during dark field microscopic examination, illumination light often enters the objective lens due to some reasons, making the background of the field of view not dark enough, resulting in a decrease in the quality of the microscopic examination. At this time, adjust the size of the aperture to darken the background, make the inspected object brighter, and enhance the microscope inspection effect. (c) Phase contrast objective: This objective is a special objective for phase contrast microscopy. Its characteristic is that a phase plate is installed at the rear focal plane of the objective. (d) No cover objective. Some objects to be inspected, such as smeared films, cannot be covered with a cover glass. Therefore, a coverless objective should be used during microscopic examination, otherwise the image quality will be significantly reduced, especially It is more obvious during high-power microscopy. The outer shell of this kind of objective lens is often marked with NC. At the same time, there is no word 0.17 on the thickness of the cover glass, but "0" is marked on it. (e) Long working distance objective lens This objective lens is a special objective lens for inverted microscopes. It is designed to meet the needs of microscopic examination of tissue culture, suspension and other materials. (5) Eyepiece The function of the eyepiece is to magnify the real image (intermediate image) magnified by the objective lens by one level and reflect the object image into the eyes of the observer. In essence, the eyepiece is a magnifying glass. It is known that the resolution capability of a microscope is determined by the numerical aperture of the objective lens, while the eyepiece only plays a magnifying role. Therefore, for structures that cannot be distinguished by the objective lens, no matter how large the eyepiece is, they still cannot be distinguished. (6) Condenser The condenser is installed below the stage. Small microscopes often do not have a condenser. When using an objective lens with a numerical aperture of 0.40 or above, a condenser is required. The condenser can not only make up for the lack of light and appropriately change the properties of the light emitted from the light source, but also focus the light on the object to be inspected to obtain the best lighting effect. There are many types of condenser structures, and depending on the numerical aperture of the objective lens, the requirements for the condenser are also different. 1． Abbe condenser This was designed by Ernst Abbe, a master of the German Optical University. The Abbe condenser consists of two lenses and has good light-gathering ability. However, when the numerical aperture of the objective lens is higher than 0.60, chromatic aberration and spherical aberration will appear. Therefore, it is mostly used on ordinary microscopes. 2. Achromatic aplanatic condenser This condenser is also known as "aspherical condenser" and "Qiming condenser". It consists of a series of lenses. It has a high degree of correction of chromatic aberration and spherical aberration and can obtain ideal images. , is the highest quality condenser in bright field microscopy, with an NA value of 1.4. Therefore, advanced research microscopes are often equipped with such condensers. It is not suitable for low-magnification objectives below 4X, otherwise the illumination source cannot fill the entire field of view. 3. Swing out condenser When using a low-magnification objective lens (such as 4X), due to the large field of view, the light cone formed by the light source cannot fill the entire field of view, causing the edges of the field of view to be dark and only the central part to be illuminated. Bright. To fill the field of view with illumination, the upper lens of the condenser needs to be rocked out of the optical path. 4. Other condensers In addition to the above-mentioned types of condensers used in bright fields, there are also condensers for special purposes. Such as darkfield condenser, phase contrast condenser, polarizing condenser, differential interference condenser, etc. The above condensers are suitable for corresponding observation methods. (7) Illumination method The illumination method of the microscope can be divided into two categories: "transmission illumination" and "epi-illumination" according to the formation of the illumination beam. The former is suitable for transparent or translucent objects to be inspected, and most biological microscopes belong to this type of lighting method; the latter is suitable for non-transparent objects to be inspected, and the light source comes from above, also known as "reflective lighting". Main applications Compared with metallographic microscopy or fluorescence microscopy. 1. Transmission illumination biological microscopes are mostly used to observe transparent specimens and need to be illuminated by transmitted light. There are two lighting methods (1) Critical illumination. After the light source passes through the condenser, the image is formed. on the object plane, as shown in Figure 5. If the loss of light energy is ignored, the brightness of the light source image is the same as the light source itself. Therefore, this method is equivalent to placing the light source on the object plane. Obviously, in critical illumination, if If the surface brightness of the light source is uneven, or there are obvious small structures, such as filaments, it will seriously affect the observation effect of the microscope. This is a shortcoming of critical lighting. The remedy is to place opal and heat-absorbing filters in front of the light source. The color film makes the illumination more uniform and avoids damage to the object being inspected due to long-term exposure of the light source. When illuminating with transmitted light, the aperture angle of the objective lens imaging beam is determined by the aperture angle of the condenser mirror image square beam. In order to make the objective lens To make full use of the numerical aperture, the condenser should have the same or slightly larger numerical aperture as the objective lens. (2) The shortcomings of uneven illumination of the object surface in Korah illumination can be eliminated between the light source 1 and the condenser. Add an auxiliary condenser 2 between 5, as shown in Figure 6.

It can be seen that since the light source is not directly used, but the auxiliary condenser 2 (also called a Cora lens) uniformly illuminated by the light source is imaged on the specimen 6, the field of view (specimen) of the objective lens is uniformly illuminated. 2. When observing opaque objects with epi-illumination, such as observing metal grinding discs through a metallographic microscope, illumination is often used from the side or from above. At this time, there is no cover glass on the surface of the object being observed, and the specimen image is generated by reflected or scattered light entering the objective lens. As shown in Figure 7. 3. Illumination method for observing particles using dark field. Ultramicroscopic particles can be observed using dark field method. The so-called ultramicroscopic particles refer to those tiny particles that are smaller than the resolution limit of the microscope. The principle of darkfield illumination is: the main illumination light is not allowed to enter the objective lens, and only the light scattered by particles can enter the objective lens for imaging. Therefore, the image of bright particles is given on a dark background. Although the background of the field of view is dark, the contrast (contrast) is very good, which can improve the resolution. Dark field lighting can be divided into one-way and two-way lighting (1) One-way dark field lighting Figure 8 is a schematic diagram of one-way dark field lighting. It can be seen from the figure that after the light emitted by the illuminator 2 is reflected by the opaque specimen piece 1, the main light does not enter the objective lens 3. The light entering the objective lens is mainly the light scattered by particles or uneven details. Obviously, this kind of one-way dark field illumination is effective for observing the existence and movement of particles, but it is not effective for reproducing object details, that is, there is a phenomenon of "distortion". (2) Bidirectional dark field illumination Bidirectional dark field illumination can eliminate the distortion shortcomings caused by one direction. In front of an ordinary three-lens condenser, an annular diaphragm is placed, as shown in Figure 9, to achieve two-way dark field illumination. The space between the last piece of the condenser and the slide glass is immersed in liquid, while the space between the cover glass and the objective lens is dry. As a result, the ring-shaped light beam passing through the condenser is totally reflected in the cover glass and cannot enter the objective lens, forming a loop as shown in the figure. Only the light scattered by the particles on the specimen enters the objective lens, forming two-way dark field illumination. 4. The composition and structure of an optical microscope. An optical microscope includes two major parts: an optical system and a mechanical device. A digital microscope also includes a digital camera system, which are described below: (1) Mechanical device 1. Frame The main part of the microscope, including the base and the curved arm. 2. The eyepiece tube is located above the frame, fixed to the frame by a circular dovetail groove, and the eyepiece is inserted into it. According to whether there is a camera function, it can be divided into binocular tube and trinocular tube; according to the adjustment method of interpupillary distance, it can be divided into hinge type and translation type. 3. The nosepiece is a rotating disk with 3 to 5 holes, respectively equipped with low- or high-power objective lenses. Rotating the nosepiece converter allows objective lenses of different magnifications to enter the working optical path. 4． The stage is a platform on which slides are placed, with a light hole in the center. There is a flexible specimen holder on the table to hold the slide. There is a moving handle on the lower right side so that the loading table can be moved in both X and Y directions. 5． Focusing mechanism: The focusing handwheel can be used to drive the focusing mechanism, causing the stage to perform coarse and fine adjustment lifting movements, so that the observed object can be focused and clearly imaged. 6. Concentrator adjustment mechanism The condenser is installed on it, and the adjustment screw can raise and lower the condenser to adjust the intensity of the light. (2) Optical system 1. Eyepiece It is a lens inserted at the top of the eyepiece tube. It consists of a set of lenses that can enable the objective lens to resolve and magnify the object image multiple times, such as 10X, 15X, etc. According to the size of the field of view that can be seen, eyepieces can be divided into two categories: ordinary eyepieces with a smaller field of view, and large-field eyepieces (or wide-angle eyepieces) with a larger field of view. The eyepieces of higher-end microscopes are also equipped with a diopter adjustment mechanism, so that the operator can easily and quickly adjust the diopter for the left and right eyes respectively; in addition, a measuring reticle can be installed on these eyepieces to measure the image of the reticle. The eyepieces can be clearly focused on the focal plane of the specimen; and, to prevent the eyepieces from being removed and reduce the possibility of damage during transportation, these eyepieces can be locked. 2. The objective lens is mounted on the hole of the converter and is also composed of a set of lenses that can clearly magnify objects. The objective lens is engraved with magnification, mainly 10X, 40X, 60X, 100X, etc. Liquid immersion objectives are often used in high-magnification objectives, that is, a liquid (such as fir oil) with a refractive index of about 1.5 is filled between the lower surface of the objective lens and the upper surface of the specimen. It can significantly improve the resolution of microscopic observation. 3. Light sources include halogen lamps, tungsten lamps, mercury lamps, fluorescent lamps, metal halide lamps, etc. 4． Concentrator includes condenser lens and aperture diaphragm. The condenser is composed of a lens, which can concentrate the transmitted light so that more light energy can be concentrated on the part being observed. The aperture diaphragm can control the light transmission range of the condenser to adjust the intensity of light.

(3) Digital camera system 1. Camera 2. Image capture card 3. Software 4. Microcomputer 5. Classification of Optical Microscopes Optical microscopes can be classified in many ways: according to the number of eyepieces used, they can be divided into binocular and monocular microscopes; according to whether the image has a three-dimensional sense, they can be divided into stereoscopic vision and non-stereoscopic vision microscopes; according to the observation object Images can be divided into biological and metallographic microscopes; according to optical principles, they can be divided into polarization, phase contrast and differential interference contrast?/ca>