Transmission electron microscope (TEM) can see the fine structure less than 0.2um that can't be seen clearly under optical microscope. These structures are called submicrostructures or ultrastructures. In order to see these structures clearly, we must choose a light source with shorter wavelength to improve the resolution of the microscope. At present, the resolution of TEM can reach 0.2nm, and the imaging principle of electron microscope and optical microscope is basically the same, except that the former uses electron beam as light source and electromagnetic field as lens.
Because the penetration of electron beam is very weak, the specimen used for electron microscope must be made into ultra-thin sections with a thickness of about 50nm. This kind of slice needs to be made with an ultra-thin slicer. The magnification of electron microscope can reach nearly one million times, and it consists of five parts: illumination system, imaging system, vacuum system, recording system and power supply system.
Type of tem:
1. Large-scale transmission electron microscope: Large-scale transmission electron microscope generally uses 80-300kV electron beam acceleration voltage, and different models correspond to different electron beam acceleration voltages, and its resolution is related to the electron beam acceleration voltage, which can reach 0.2-0. 1nm. High-end models can achieve atomic resolution.
2. Low-voltage transmission electron microscope: The electron beam acceleration voltage (5kV) used by low-voltage small transmission electron microscope is much lower than that of large transmission electron microscope. The lower accelerating voltage will enhance the intensity of the interaction between the electron beam and the sample, thus improving the contrast and contrast of the image, which is especially suitable for polymer, biological and other samples. At the same time, the low voltage transmission electron microscope has little damage to the sample.
3. Freezing Electron Microscope: Freezing Electron Microscope usually adds a sample freezing device to the common transmission electron microscope to cool the sample to the temperature of liquid nitrogen, which is used to observe temperature-sensitive samples such as protein and biological slices. By freezing the sample, the damage of the electron beam to the sample can be reduced, and the deformation of the sample can be reduced, so that a more realistic sample morphology can be obtained.
Transmission electron microscope imaging principle transmission electron microscope, that is, transmission electron microscope is a kind of electron microscope. Electron microscope is a high-precision electron optical instrument with high resolution and magnification, and it is an important tool for observing and studying the microstructure of matter.
Electron microscope is an instrument that uses electron beam and electron lens instead of light beam and optical lens according to the principle of electron optics, so that the fine structure of matter can be imaged at a very high magnification. The resolving power of an electron microscope is expressed by the minimum distance between two adjacent points that it can resolve. In 1970s, the resolution of transmission electron microscope was about 0.3 nm (human eye resolution was about 0. 1 mm). At present, the maximum magnification of electron microscope is more than 3 million times, while the maximum magnification of optical microscope is about 2000 times, so some heavy metal atoms and ordered atomic lattices in crystals can be directly observed through electron microscope.
In 193 1, Knohl and ruska in Germany modified a high-voltage oscilloscope with a cold cathode discharge electron source and three electronic lenses, and obtained an image magnified more than ten times, which confirmed the possibility of magnifying imaging by an electron microscope. 1932, after ruska's improvement, the resolution of the electron microscope reached 50 nanometers, which was about ten times that of the optical microscope at that time, so people began to pay attention to the electron microscope. In the 1940s, Hill in the United States used astigmatism device to compensate the rotational asymmetry of electronic lens, which made a new breakthrough in the resolution of electron microscope and gradually reached the modern level. In China, a transmission electron microscope was successfully developed at 1958 with a resolution of 3 nm, and a large electron microscope with a resolution of 0.3 nm was manufactured at 1979.
Although the resolution of the electron microscope is far superior to that of the optical microscope, it is difficult to observe the living body because the electron microscope needs to work in a vacuum, and the irradiation of the electron beam will also damage the biological sample. Other problems, such as the brightness of electron gun and the improvement of the quality of electronic lens, need further study.
The imaging principle of transmission electron microscope is that the electron beam with a certain aperture angle and intensity provided by the illumination part is projected on the sample on the object surface of the objective lens in parallel, and the electron beam passing through the sample and the objective lens forms the maximum diffraction amplitude on the back focal plane of the objective lens, that is, the first diffraction spectrum. These diffracted beams interfere with each other on the image plane of the objective lens to form a first electronic image reflecting the characteristics of the sample micro-area. By focusing (adjusting the excitation current of the objective lens), the image plane of the objective lens is consistent with the object plane of the intermediate mirror, the image plane of the intermediate mirror is consistent with the object plane of the projection mirror, and the image plane of the projection mirror is consistent with the fluorescent screen, so that an electronic image with a certain contrast and magnification after being amplified by the objective lens, the intermediate mirror and the projection mirror can be observed on the fluorescent screen. Because the thickness, atomic number, crystal structure or crystal orientation of each micro-area of the sample are different, the electron beam intensity passing through the sample and the objective lens is also different, so a microelectronic image of the characteristics of the micro-area of the sample reflected by the difference of light and dark appears on the fluorescent screen. The magnification of electronic image is the product of the magnification of objective lens, intermediate mirror and projection mirror.
Imaging principle of electron microscope. The imaging principle of transmission electron microscope can be divided into three situations:
1. absorption image: When electrons impact high-quality and high-density samples, the main phase-forming effect is scattering. The place with large mass thickness on the sample has a large scattering angle for electrons, less electrons pass through it, and the image brightness is dark. The early transmission electron microscope was based on this principle.
2. Diffraction image: After the electron beam is diffracted by the sample, the amplitude distribution of diffracted waves at different positions of the sample corresponds to the different diffraction abilities of each part of the crystal in the sample. When crystal defects appear, the diffraction ability of the defect part is different from that of the complete area, which makes the amplitude distribution of diffraction wave uneven and reflects the distribution of crystal defects.
3. Phase image: When the sample is thinner than 100_, electrons can pass through the sample, and the amplitude change of the wave can be ignored, and the imaging comes from the phase change.
Second, the principle of scanning electron microscope imaging
A scanning electron microscope generates an image of the sample surface by scanning the sample surface with a focused electron beam.
Electrons interact with atoms in the sample to generate various signals including the surface morphology and composition information of the sample. The electron beam is usually scanned in a raster scanning mode, and the position of the electron beam is combined with the detected signal to generate an image.
The resolution of scanning electron microscope is better than 65438±0nm. The samples can be observed under high vacuum, low vacuum and humid conditions (using environmental scanning electron microscope) and a wide range of low or high temperatures.
The most common scanning electron microscope mode is to detect the secondary electrons emitted by atoms excited by electron beams. The number of secondary electrons that can be detected depends on the morphology of the sample and other factors.
By scanning the sample and collecting the emitted secondary electrons with a special detector, an image showing the surface morphology can be created. It can also produce a high-resolution image of the sample surface, and the image is three-dimensional, identifying the surface structure of the sample.
Extended data:
Biological samples must be pretreated before being observed by perspective electron microscope. With the needs of different research requirements, scientists adopt different treatment methods.
1. fixation: In order to preserve the sample as much as possible, glutaraldehyde is used to harden the sample, and osmic acid is used to dye the fat.
2. Cold fixation: put the sample into liquid ethane for quick freezing, so that water will not crystallize to form amorphous ice. The sample stored in this way has less damage, but the contrast of the image is very low.
3. Dehydration: replace water with ethanol and acetone.
4. Buffering: the sample can be divided after buffering.
5. Segmentation: Slice the sample with a diamond blade.
6. Dyeing: Heavy atoms such as lead or uranium have higher ability to scatter electrons than light atoms, so they can be used to improve contrast.
Transmission electron microscope transmission electron microscope, referred to as transmission electron microscope, is a kind of microscope that uses high-speed electron beam as light source, penetrates solid samples, and then images through electromagnetic lens.
Transmission electron microscope consists of electron optical system, observation and recording system, vacuum and cooling system and power supply system. The electro-optical system can be divided into two parts: the illumination system and the imaging system, which are placed in the vacuum lens barrel together with the observation and recording system. The sample table is located between the illumination system and the imaging system (Figure 5-3).
Figure 5-3 Structure Diagram of Transmission Electron Microscope
(According to JEOL Corporation of Japan)
The imaging principle of transmission electron microscope is similar to that of optical microscope, and its image is a contrast image with different brightness formed by different transmission electron densities on the imaging plane. This density difference can be observed through the conversion of fluorescent screen or photographic negative. According to different contrast sources, transmission electron microscope images can be divided into four types: thick contrast image, diffraction contrast image, phase contrast image and Z contrast image. Limited by space, this section briefly introduces commonly used contrast images of mass and thickness and diffraction contrast images.
The contrast of quality and thickness contrast images is caused by the difference of sample quality and thickness. It is suitable for observing amorphous samples, such as carbon black. Diffraction contrast image, abbreviated as diffraction contrast image. The contrast is caused by the difference of Bragg diffraction conditions in different parts of the sample, which reflects the difference of diffraction intensity of incident electrons in different parts of the sample. Contrast images can be divided into bright-field images and dark-field images. Bright field image (abbreviated as BFI) is imaged by transmitted light beam, which forms a dark image on a bright background (Figure 5-4). Dark-field imaging (abbreviated as DFI) only uses a diffracted beam to form a bright image on a dark background. Because diffraction contrast is closely related to diffraction conditions, it is very sensitive to the orientation change of diffraction network in crystals, so it is a powerful means to study crystal defects.
For a long time, the images of transmission electron microscope have been observed through the fluorescent screen in the observation room and recorded with photographic film. In recent years, CCD camera can be equipped at the position of photographic film to digitize the image and facilitate computer storage.
Figure 5-4 Bright-field image of olivine dislocation in Taizhou meteorite
(Provided by Zhang Fusheng)
The most outstanding advantages of transmission electron microscope are high image resolution and large effective magnification. Its point resolution (the shortest distance between two distinguishable points in an image) is about 0. 17 ~ 0.20 nm, and its lattice resolution (the shortest distance between stripes in a lattice fringe image) is 0. 1 ~ 0. 14 nm. The resolution of transmission electron microscope corrected by spherical aberration is 0.08nm, which can be enlarged by 654.38 0 million times, and almost all the atoms in the crystal can be distinguished.
Another feature of transmission electron microscope is that an electron diffraction pattern can be obtained by inserting a selective aperture in the imaging system, and the structure can be analyzed in situ while observing the image (see section 4 of this chapter). The principles of electron diffraction and X-ray diffraction are basically the same, and the diffraction patterns obtained are also very similar.
The basic requirements of transmission electron microscope for the sample are as follows: ① In order to make the electron beam penetrate the sample, its thickness should be below 100nm; ② In the process of sample preparation, the ultrastructure of the sample must be well preserved, and the structure and properties of the sample should be strictly prevented from changing and the sample should be polluted. ③ The sample should be firmly placed on a special copper net with a diameter of 3mm, so that it can withstand the bombardment of electron beams and prevent mechanical vibration during loading and unloading; ④ The sample must be conductive. For non-conductive samples, a thin carbon film should be sprayed on them. For geological samples, they are usually ground into thin slices and observed under polarizing microscope. Select the part that needs further study, cut it off, stick it on the copper mesh, and then thin it in the ion thinning instrument until it is partially perforated, and the edge part can be observed under the transmission electron microscope.
Transmission electron microscope equipped with X-ray energy spectrometer can observe the image in situ and analyze the element composition of micro-area.
Principle of electron microscope The principle of electron microscope is as follows:
A, transmission electron microscope
Transmission electron microscope, usually called electron microscope or electron microscope, is the most widely used electron microscope.
1. working principle: under vacuum condition, after being accelerated by high voltage, the electron beam penetrates the sample to form scattered electrons and transmitted electrons, which are imaged on the fluorescent screen under the action of electromagnetic lens. When the electron beam is projected on the sample, it can emit electrons with different density of tissue components. For example, when an electron beam is projected on a high-quality structure, electrons are scattered more, so fewer electrons are projected on the fluorescent screen, showing a dark image, while an electronic photograph is black.
2. Main advantages: high resolution, which can be used to observe the ultrastructure of tissues and cells and the panorama of microorganisms and biomacromolecules.
Second, the scanning electron microscope
Scanning electron microscope (SEM) is mainly used to observe the surface morphology, cross-sectional structure and inner surface structure of the lumen.
1. Working principle: SEM uses secondary electronic signal imaging to observe the surface morphology of the sample. Scanning the sample surface with a very fine electron beam excites the sample surface to emit secondary electrons, and the generated secondary electrons are collected by a special detector to form an electrical signal and sent to a picture tube to display the object on the screen. A three-dimensional image of the surface of a cell or tissue that can be photographed.
2. Main advantages: the depth of field is long, and the obtained image has a strong stereoscopic effect, which can be used to observe various morphological characteristics of biological samples.
What is a transmission electron microscope? Transmission electron microscope (TEM) projects an accelerated and concentrated electron beam onto a very thin sample, and the electrons collide with atoms in the sample to change direction, thus producing solid angular scattering. The scattering angle is related to the density and thickness of the sample, so images with different light and dark can be formed. Generally, the resolution of transmission electron microscope is 0. 1 ~ 0.2 nm, and the magnification is tens of thousands to millions of times, which is suitable for observing ultrastructure. Transmission electron microscope is widely used in materials science and biology. Because electrons are easily scattered or absorbed by objects, the penetration is low, and the density and thickness of the sample will affect the final imaging quality. Need to prepare thinner ultra-thin slices, generally 50 ~ 100 nm. Therefore, the samples observed by transmission electron microscope need to be treated very thin. Commonly used methods are: ultra-thin slicing method, frozen ultra-thin slicing method, frozen etching method, frozen fracture method and so on.