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microscope
microscope
An instrument or equipment that magnifies a tiny object or a tiny part of an object at a high magnification for observation. It is widely used in industrial and agricultural production and scientific research. Biology and medical workers often use microscopes in their work. It can be roughly divided into optical microscope and electron microscope.
Optical microscope is a microscope with visible light as its light source. Ordinary optical microscope can be divided into two parts: optical system and mechanical device. The optical system mainly includes eyepiece, objective lens, condenser, diaphragm and light source. The mechanical device mainly includes lens barrel, lens column, objective table, lens seat, thickness adjusting screw, etc. (Figure 1 [optical microscope]). Its basic optical principle is shown in Figure 2 [Imaging Principle Mode Diagram of Optical Microscope], in which the small convex lens on the left represents a group of lenses with short focal length, which is called objective lens. The large convex lens on the right represents a lens with a long focal length, which is called an eyepiece. The observed object (AB) is located slightly outside the focal point (F) of the objective lens. After passing through the objective lens, the light of the object forms an inverted magnified real image (BA) slightly inside the focal point (F) of the eyepiece. The observer's eyes further enlarge the real image (BA) into an inverted virtual image (BA) through the eyepiece.
The eyepiece is located above the microscope barrel and usually consists of two convex lenses. In addition to further expanding the real image formed by the objective lens, it also limits the field of vision observed by the eyes. According to the magnification, there are three kinds of commonly used eyepieces: 5 times, 10 times and 15 times.
The objective lens is usually located under the microscope barrel, close to the observed object. It consists of 8 ~ 10 lenses. Its function is to enlarge (produce an enlarged real image of an object), ensure the image quality and improve the resolution. Ordinary objective lenses can be divided into low magnification (4 times), medium magnification (10 or 20 times), high magnification (40 times) and oil-immersed objective lenses (100 times). A plurality of objective lenses are embedded in the mirror changing turntable, and the turntable can be rotated as required to select objective lenses with different multiples.
The magnification of microscope is the multiple of eyepiece multiplied by the multiple of objective lens. If the eyepiece is 10 times and the objective is 40 times, the magnification is 40× 10 times (the magnification is 400 times). An excellent microscope can magnify 2000 times and distinguish two points which are 1× 10cm apart.
When white light passes through a convex lens, the shorter wavelength light (blue-purple) refracts more than the longer wavelength light (red-orange). Therefore, when imaging, various spectra appear around the image, with a blue or red glow. This color defect is called chromatic aberration. Because the angles of light entering (leaving) the mirror surface of the lens are different, the refraction angle of light passing around the lens is greater than that passing through the center of the lens. Therefore, in the process of imaging, blurred and distorted images will appear around the image. The defect of this imaging curved surface is called spherical aberration. A series of convex and concave lens groups with different shapes, structures and distances cooperate with each other, which can correct chromatic aberration and spherical aberration to the maximum extent and form bright, clear and accurate images. This is why the eyepiece or the objective lens is composed of a group of lenses respectively. This kind of lens is called flat-field achromatic lens.
When light is projected from one medium (such as air) into another medium with higher density (such as glass), it will bend to the "normal" (the line perpendicular to the interface of the medium), as shown in Figure 3 [when light passes through the objective lens]. Light will deviate from the "normal" when it enters the non-dense medium (air) from the dense medium (glass). For example, when the AOB line (Figure 3a) enters the air through the condenser (refractive index: 1.5 1), it will also be deflected and refracted outward, so the amount of light entering the objective lens is greatly reduced, and the resolution of the image is also reduced. When an objective lens with 100 times is used, if oil is filled between the objective lens and the cover glass (the refractive index is also 1.5 1) to isolate the air, the light can almost enter the objective lens without refraction, which increases the brightness and resolution of the image. This objective is called an oil-immersed objective (Figure 3b).
The condenser is located under the microscope stage, which can collect the light from the eye source and concentrate the light on the specimen, so that the specimen is evenly irradiated with moderate light intensity. The lower end of the condenser is equipped with an aperture stop (aperture) to control the thickness of the light beam.
The light source of ordinary optical microscope is located under the condenser, which is a special strong light bulb with uniform illumination and variable resistance, which can change the intensity of light.
Because the light emitted by the light source of an ordinary optical microscope is transmitted upward from the lower part of the mirror, passes through the condenser and the objective lens, and reaches the eyepiece, it is necessary to cut the observed sample into translucent sheets with a thickness of about 6m and dye them to show different fine structures such as tissues and cells. The whole treatment process is called conventional tissue sectioning technology, including selecting appropriate tissue materials, fixing them with formaldehyde (formalin) solution, gradually dehydrating them with alcohol, embedding them in paraffin, slicing and fixing the tissues on a glass with a slicer, then dyeing them with hematoxylin-eosin dye, and finally sealing the tissue sections with optical resin glue. The prepared tissue sections can be stored for a long time.
The eyepiece and objective lens of the microscope are installed at both ends of the lens barrel, and their distance is fixed. Put the tissue slice on the stage, and turn the coarse adjustment screw to make the stage close to the objective lens. The tissue slice enters the first focal plane of the objective lens, and the tissue image in the specimen can be seen in the eyepiece. Then use the fine-tuning screw to make the image in the eyepiece clear and easy to observe. When changing the magnification, it is necessary to change the eyepiece or objective lens.
Optical microscopes commonly used in medicine and biology are as follows 12:
The dark field microscope is equipped with a dark field condenser under the ordinary optical microscope (Figure 4), and the light emitted by the light source below is reflected by the parabolic condenser to form a strong beam that passes through the field of view of the microscope without entering the objective lens. Therefore, the field of vision is dark, and the size and shape of scattered light from particles with a diameter greater than 0.3m in the field of vision can be clearly seen. You can even see a few nanometers of particles that ordinary bright-field microscopes can't see. Therefore, it is often used for the biopsy of some bacteria and cells.
The solid-state microscope consists of binocular eyepiece and objective lens. The magnification is 7 ~ 80 times. Illuminate above or below the side with a microscope lamp. An upright enlarged real image is formed in the eyepiece, which can be used to observe the three-dimensional shape, color and surface microstructure of unprocessed objects, perform micro-dissection operations, and also observe the tissue sections of organisms.
Under the irradiation of short wavelength light waves (ultraviolet light or violet-blue light, wavelength 250 ~ 400 nm), some substances absorb light energy, are excited and release longer light waves (blue, green, yellow or red light, wavelength 400 ~ 800 nm) with degraded energy, which is called fluorescence. A substance will fluoresce under short wave irradiation. For example, most lipids and protein in tissues can emit light blue fluorescence after irradiation, which is called autofluorescence. But most substances need to be dyed with fluorescent dyes (such as acridine orange, fluorescein isothiocyanate, etc. ) can emit fluorescence under short light wave irradiation. The light source of fluorescence microscope is high-pressure mercury lamp. The ultraviolet light emitted by the fluorescence microscope is filtered by the excitation filter (which can appropriately excite the fluorescent substances in the sample), and then emitted to the Proom dichroic mirror to reflect the excitation light downwards, and then projected onto the sample dyed with fluorescent dyes through the objective lens. When the dye is excited, it emits fluorescence, which can be observed through objective lens, dichroic mirror and eyepiece. A blocking filter is placed under the eyepiece (only fluorescence with a specific wavelength is allowed to pass through) to protect the eyes and reduce the darkness of the field of vision (Figure 4 [Optical principle of fluorescence microscope]). Fluorescence microscope is characterized by high sensitivity. In dark field, low-concentration fluorescent staining can show the existence of samples, and its contrast is about 100 times that of visible light microscope. In 1930s, fluorescence staining was used to observe and study the morphology of microorganisms, cells and fibers such as bacteria and molds. For example, acid-fast fluorescent staining can help to find mycobacterium tuberculosis in sputum. The technique of labeling protein with fluorescent dyes came into being in the 1940s. This technique has been widely used in the routine technique of immunofluorescent antibody staining, which can detect and locate the antigens and antibodies of viruses, bacteria, molds, protozoa, parasites, animals and human tissues, and can be used to explore the etiology and pathogenesis, such as the classification and diagnosis of glomerular diseases, the relationship between papillomavirus and cervical cancer, etc. Widely used in medical experimental research and disease diagnosis.
When the light emitted by the polarizing microscope from the light source passes through air and ordinary glass, it vibrates in all directions with the same amplitude in the plane perpendicular to the light and quickly propagates to the front. This is the wave principle of light. Air and ordinary glass are isotropic bodies, also known as single refraction. If the light from a light source passes through an anisotropic body (also known as a birefringent body), it will split a beam of light into two beams with only one vibration plane and vertical vibration direction. The vibration direction, speed, refractive index and wavelength of these two beams of light are different. In this way, light with only one vibration plane is called polarized light. Polarizing microscope is designed by using this phenomenon. In the polarizing microscope, an analysis lens is inserted between the objective lens and the eyepiece, and a polarizing lens is inserted between the light source and the condenser, so that the circular stage can rotate 360 degrees (Figure 5 [Optical Principle of Polarizing Microscope]). When the polarizing and analyzing lenses are in orthogonal positions, the field of view is completely black. Place the inspected object on the microscope workbench. If the object is single refraction, rotate the stage and the field of view will always be dark. If the object in the field of vision is bright and dark after rotating the stage for one week, it means that the object is birefringent. Uric acid salt crystals in many crystalline substances (such as gout nodules, urinary calculi and gallstones). ), elastic fibers, collagen fibers, chromosomes and amyloid fibrils in human tissues, etc. It is birefringent and can be analyzed qualitatively and quantitatively by polarization microscope technology.
Phase microscope is also called phase contrast microscope or phase contrast microscope. The reason why ordinary optical microscope can't see the images of unstained tissues, cells, bacteria, viruses and other living things is because the difference (contrast) of light passing through the sample is very small. After the specimen is dyed, the amplitude (brightness) and wavelength (color) change, which affects the contrast and obtains the image. However, dyeing will lead to sample deformation and biological death. It is necessary to use a phase microscope to observe fresh unstained tissues, cells or other tiny life forms. The principle of phase microscope is that two light waves interfere with each other because of the phase difference, and the intensity and contrast of light waves change to form a visible image. The light emitted by a point light source can be displayed as a sine wave pattern (Figure 6a [Phase Microscope]). The distance between the two peaks is the wavelength, and the amplitude of the wave indicates the brightness of light (large amplitude, high brightness). Imagine that two light waves emitted by the same light source pass through air and a transparent medium at the same time. When passing through a transparent medium with a certain thickness, the speed of light waves will decrease, but the brightness of light will not change. After the light wave passes through the transparent medium, it lags behind another light wave that has been advancing in the air, so the two light waves have a phase change (phase difference). But the human eye can't tell the phase difference between these two parallel rays. If these two light waves hit the same point on the screen, one light wave lags behind the other by half a wavelength, that is, the two light waves interfere and cancel each other because of their opposite phases, and the light disappears, or the relative amplitudes affect each other and the light weakens. If one light wave is delayed by one wavelength, but the two light waves are in the same phase, then the light will be enhanced by the superposition of the waves.
The basic structure of a phase microscope is the same as that of an ordinary optical microscope. The differences are as follows: ① On the objective lens, a disc-shaped phase plate is installed on the second focal plane of the objective lens (Figure 6b [Phase Microscope]). (2) Under the condenser, install an annular beam at the first focal plane of the condenser, and carve a narrow slit on the beam to let the annular strong light pass through (Figure 6c [Phase Microscope]). As shown in fig. 6d, the light emitted from point A of the annular beam becomes parallel light after passing through the condenser. When light passes through the sample on the stage, due to the different refractive index of each particle (such as point B) in the sample, interference and diffraction will occur, which are divided into unbiased wave (solid line) and polarized wave (dotted line). Unbiased waves are focused on the point A of the phase plate through the objective lens, and then are evenly distributed on the image plane of the specimen through the phase plate to become the background. After passing through the objective lens, the deflected wave bypasses the point A of the phase plate, passes through the phase plate, and is also focused on the b of the image plane. In other words, the unbiased wave and the deflected wave pass through different parts of the phase plate respectively. By coating different coatings on different areas of the phase plate, the speed and brightness of unbiased wave or biased wave can be changed respectively, so that the two light waves have a phase difference of half a wavelength or one wavelength, and their combined wave on the image plane will have a bright and dark contrast, and all details in the sample can be seen.
In a word, the phase microscope uses the difference of refractive index or thickness of particles in the sample to produce the phase difference of light, so that fresh samples can be seen without dyeing, and fine structures such as mitochondria and chromosomes in living cells can be observed. It can also be applied to the study of small organisms such as molds, bacteria, viruses, etc., and observe the biological behaviors such as specimen morphology, quantity, activity, division, reproduction, etc., and can be measured and compared.
The objective lens of the inverted microscope is oriented downward close to the specimen. The objective lens of an inverted microscope is in a vertical position, so the longitudinal axes of the eyepiece and the lens barrel make an angle of 45 degrees with the longitudinal axis of the objective lens. The stage has a large area. Above the objective lens, there is a long focal length condenser and an illumination light source. The objective lens and condenser can be equipped with accessories of phase microscope. The magnification is 16 ~ 80 times. Tissue culture bottles and Petri dishes can be placed directly on the stage to observe the morphology, quantity and dynamics of fresh specimens, living bodies and cells without staining. The results of porous micro-biochemical and immune reaction plates can be observed. The inverted microscope can be replaced by ordinary bright-field optical lens; Polarized light, differential interference difference and fluorescent accessories can be assembled and observed.
Differential interference microscope (DIC) is also called interference or interference microscope. It can see and measure tiny phase changes, similar to a phase microscope, so that colorless and transparent samples have changes in light and shade and color, thus enhancing contrast. Polarization and interference elements and a 360-degree rotating stage are installed on the basic structure of ordinary optical microscope, and the interference principle of polarized light is used. As shown in Figure 7 [Optical Principle of Differential Interference Differential Microscope], a polarizer and a beam splitter prism are arranged above the light source. The linearly polarized light from the polarizer passes through the beam splitting prism and is divided into two linearly polarized lights that vibrate perpendicularly to each other. These two rays are refracted by the condenser and directed to the sample. Because the refractive index of each particle in the sample is different, the phase of some light waves will change and shift laterally due to interference. After the two beams pass through the objective lens, they are combined by the second component prism and interfered by the analyzer. Every point in the final image is a mixed image formed by overlapping two different images of the same point on the object, so it can be recognized by the naked eye.
Differential interference microscope can also observe colorless and transparent objects that can't be seen in ordinary bright field, and can observe living bodies such as cells and bacteria. The image is stereoscopic, which is more detailed and realistic than phase microscope. It can be used to study various parts of living cells in more detail. If white light is used for illumination, various colors will appear in different phases. When the stage rotates, the color will change. Monochromatic light produces contrast between light and dark, and various components show different contrasts. Differential interference microscope can also be used as a kind of ultra-micro optical balance with high precision. The accurate mass of dry objects can be estimated as small as 1× g, and the refractive index increases by 0.00 18 with the increase of the concentration of solid matter in cells. The refractive index of each phase of the cell can be estimated according to the species difference between it and the related area (suspended area), so that the dry weight of some components in the cell can be further calculated.
Photographic Microscope Modern high-quality microscopes can be equipped with various accessories for microphotography, which can timely and completely preserve scientific data. Photographic microscope requires precise optical system and parts structure, and firm and stable mirror body. Equipped with three eyepiece tubes, two 45-degree observation eyepiece tubes and a central vertical tube are equipped with 135 camera, exposure measurement accessories, photographic eyepiece and viewfinder lens, which can be used for framing and focusing. The condenser can adjust the center of the field of view and is equipped with an aperture stop to make the field of view illuminate evenly. The mirror base is provided with an adjustable field of view diaphragm, a voltmeter and a voltage display lamp. There is a variable resistor to adjust the illumination brightness. The lighting source is 6 ~ 12V 40 ~ 100W halogen bulb. In 1980s, the automatic exposure microphotograph device had many functions, such as automatic film winding, automatic photometry, automatic exposure control, color temperature measurement and adjustment, reciprocity law failure compensation and so on. All these are automatically controlled by the computer, which can show black and white photographic films, color negative films and color slides.
The central vertical lens barrel can also be equipped with a TV camera or a 16mm movie camera and a control device, which can regularly freeze or continuously record living specimens.
The universal photographic microscope system used for research integrates the functions of ordinary bright field, dark field, polarized light, fluorescence, phase, differential interference difference and microphotography. There are also functions such as automatic focusing controlled by computer, automatic switching of objective lens, automatic matching of condenser lens and automatic adjustment of light source brightness. The fuselage is equipped with two 135 cameras and a 4×5 inch large format camera. You can also install a TV camera and a 16mm film camera. It also has many functions such as automatic film winding, automatic metering, automatic exposure control, measuring and adjusting color temperature, and reciprocal fuselage fault compensation.
The resolution of electron microscope is limited by the wavelength of light wave used. Objects smaller than the wavelength of light waves cannot be imaged because of diffraction. The resolution of the most advanced optical microscope is limited to about 200 nanometers (2000). In order to break through this limit, electron rays can be used instead of light waves. When electron particles move at high speed, their behavior is similar to the propagation process of light waves. The wavelength of a moving electron depends on its speed. When the voltage is raised to 500,000 volts, its wavelength is 0.00 1nm(0.0 1), that is, the wavelength of electron rays is about one hundred thousandth of that of visible light, the resolution is about 4, and the magnification is many orders of magnitude higher than that of the most advanced optical microscope. A microscope with electron rays as its light source is called an electron microscope. The resolution of electron microscope used in modern medicine and biology is 5 ~ 10, that is, the magnification is10 ~ 200,000 times.
Due to the different thickness of specimens, thin specimens cut by ultrathin microtome can be observed by transmission electron microscope. Specimens that cannot be cut very thin can be observed by scanning electron microscope.
Transmission electron microscope is the most commonly used electron microscope. An electron gun consists of an electron gun, an electromagnetic lens system, a fluorescent screen (or a camera), a lens barrel, a lens holder, a transformer, a voltage stabilizer, a high-voltage cable, a vacuum pump system, a console and other components, and is equivalent to a light source in an optical microscope, supplying and accelerating the electron beam emitted by the cathode hot tungsten filament. The voltage used by the electron microscope is generally 200,000 ~ 300,000 volts, which is enough to make the electrons in the electron gun fly out at high speed. Electrons pass through the condenser lens to reach the sample. Because the specimen is very thin, high-speed electrons can pass through it, and because the thickness or density of each part of the specimen is different, the electrons passing through it are also different. The voltage needs to be strictly stable to make the imaging stable, and small voltage changes will cause serious interference. The brightness of the image can be controlled by an electron gun.
The electromagnetic lens group is equivalent to the condenser, objective lens and eyepiece system in the optical lens. When the electron beam passes through the center of the circular magnetic field of each electromagnetic lens, it can be converged to produce an image. The lens system of electron microscope consists of four groups of electromagnetic lenses, including condenser lens, objective lens, intermediate lens and projection lens (eyepiece). The current of the condenser can be changed to focus the electron beam on the specimen and provide "illumination". The objective lens is close to the focus of the sample. Through the three-stage amplification of objective lens, intermediate mirror and projection mirror, a high-magnification image can be obtained at a certain distance, and the final image is projected on the screen. Black-and-white film can be used on the fluorescent screen to make a photo base. Changing the current in the electromagnetic coil makes the electromagnetic lens focus, resulting in different magnifications (Figure 8 [Transmission Electron Microscope]).
In order to minimize the chance of scattering when electrons collide with air molecules in the electron microscope, the vacuum degree in the lens barrel is very high, so the sealed lens barrel is connected with a vacuum pump. Because the specimen needs to be placed in a vacuum lens tube, it is impossible to check the living material.
Optical mirrors mainly use visible light waves as light sources. After the sample is dyed, the wavelength (color) and amplitude (brightness) of light change, which affects the contrast and obtains the image. An electron microscope uses electron rays. The penetration of electron beam is not strong, so the specimen examined by power mirror must be cut into thin slices as thin as 50 ~ 100 nm. The steps of making electron microscope sections are similar to those of light microscope sections, and they are also composed of procedures such as fixation, dehydration, embedding, slicing and dyeing. First, take out the material from the specimen to be observed, and the volume is about 1. After double fixation with glutaraldehyde and osmium tetroxide, it was dehydrated by alcohol (or acetone) step by step, embedded in epoxy resin, and sliced by ultra-thin microtome. In the electron microscope, the formation of the image is the result of different scattering of electrons from different parts of the tissue slice, and the dense parts (details) in the specimen have strong scattering. Various heavy metal salts can be used to increase the contrast, and uranium acetate and lead citrate are commonly used for double staining. Because the electron beam can't penetrate the glass, the dyed film slices are placed on a small copper grid for electron microscope observation.
Freeze-etching technology is a new specimen processing technology of electron microscope, which was invented in 1950s and improved continuously later. The main principle is that biological samples frozen rapidly in ultra-low temperature (-200℃) liquid nitrogen are crushed in a vacuum freezing device, so that the internal structures of organelles in different parts are exposed, showing three-dimensional structures with different heights. Spray a platinum-carbon film (replica) on the newly formed fracture surface. After coating, the specimen was digested in strong acid or strong alkaline corrosive solution, and the composite film floated, salvaged and cleaned, and placed on a small copper net for electron microscope observation and photography. Freeze etching technology plays an important role in the study of cell biofilm structure (such as cell membrane, mitochondria, endoplasmic reticulum, etc.). ).
A scanning electron microscope (SEM) is used to generate an electron optical image on the thick surface of the sample (Figure 9 [SEM structure diagram]). The structure principle of electron gun and electromagnetic lens of scanning electron microscope is similar to that of transmission electron microscope. A large number of electrons generated by the electron gun are continuously converged by three groups of electromagnetic lenses to form very fine electron rays (electron probes). Under the action of two pairs of deflection coils in the electron microscope tube, the electron rays are scanned on the surface of the sample in turn. Because the current from the sawtooth wave generator is supplied to two groups of deflection coils in the lens barrel and display tube of the electron microscope at the same time, the electron rays of the display generate synchronous scanning on the screen. Electrons emitted from the sample are collected by the detector, amplified by the video amplifier, and the brightness of the display tube is controlled. Therefore, the brightness of scanning on the fluorescent screen is controlled by the number of electrons generated at corresponding points on the surface of the sample, thereby displaying a high magnification image of the sample on the fluorescent screen. By controlling the currents of two groups of deflection coils, the magnification can be controlled. In addition, the same synchronous scanning display tube for photography is installed.
In the preparation of SEM specimens, it is necessary to dehydrate and basically keep their natural state, so the critical freeze-drying technology of specimens is adopted: tissue surface cleaning, glutaraldehyde and osmium tetroxide fixation, and acetone dehydration step by step. Because amyl acetate is very easy to replace liquefied Co, gradient amyl acetate is used to replace acetone first. Then put the sample into a closed pressure chamber and introduce liquid Co to immerse the sample. Carbon monoxide quickly completely replaced amyl acetate in the sample and discharged the latter from the pressure chamber. At the same time, the mutual transformation between liquid Co and rapidly evaporating gaseous Co molecules in the pressure chamber reaches a dynamic balance. When the temperature increases gradually, the evaporation of liquid Co accelerates and the density decreases accordingly. When the critical temperature of Co reaches 3 1. 1℃, the gas-liquid density is the same, and the difference between the two phases disappears completely, that is, the phase equilibrium is reached, and the surface tension is zero. Keep the temperature slightly higher than the critical temperature, and slowly discharge carbon monoxide gas. When the carbon monoxide is exhausted, the sample will be dried. Take out the dried specimen, spray a layer of carbon alloy in vacuum, or put it in an ion plating machine for platinum and gold plating, so as to increase the conductivity, contrast and stability of the specimen. Then it can be observed by scanning electron microscope.
Scanning electron microscope (SEM) has the advantages of high resolution, long depth of field, wide field of view, three-dimensional structure display, easy observation and simple sample preparation. It is increasingly used in biology and medicine to observe and study the expression form and internal three-dimensional structure of biological specimens. The resolution of scanning electron microscope has reached 70, and the molecular structure of deoxyribonucleic acid (DNA) can be directly observed.