This paper discusses the development status and history of electron microscope, introduces the structures, principles and applications of several advanced electron microscopes in the field of biology, and discusses their applications in histological research. Keywords: electron microscope; Introduction to histological research: Microscopy is a microscopic analysis technique specially used for chemical composition analysis, microscopic morphology observation and microstructure determination in tiny areas of matter. In 1930s, the invention of transmission electron microscope marked the birth of electron microscopy, and people can further study the ultrastructure of matter. On the basis of ordinary optical microscopy, electron microscopy has further broadened people's observation horizons, played an important role in various fields and been widely used in scientific fields. In the field of biological research, electron microscopy has promoted the development of histology, cell biology, molecular biology and other disciplines, so it has an irreplaceable lofty position.

I. Electron Microscope Technology

1. 1 The definition and composition of electron microscope, referred to as electron microscope for short, is an instrument that uses electron beams and electron lenses instead of light beams and optical lenses according to the principle of electron optics, so that the fine structure of matter can be imaged at a very high magnification. [1] An electron microscope consists of three parts: a lens barrel, a vacuum device and a power cabinet. The lens barrel is mainly composed of an electron source, an electron lens, a sample holder, a fluorescent screen and a detector, which are usually assembled in a row from top to bottom. ① Electron lens: used to focus electrons, which is the most important part in the lens barrel of electron microscope. Generally, magnetic lenses are used, and sometimes electrostatic lenses are used. It uses a spatial electric field or magnetic field symmetrical to the axis of the lens barrel to bend the electron trajectory to the axis to form a focus, and its function is the same as that of an optical lens (convex lens) in an optical microscope, so it is called an electronic lens. The focal length of the optical lens is fixed, while the focal length of the electronic lens can be adjusted, so the electronic microscope does not have a movable lens system like the optical microscope. Most modern electron microscopes use electromagnetic lenses, and very stable DC excitation current focuses electrons through the strong magnetic field generated by coils with pole shoes. ② Electron source: It consists of a cathode for releasing free electrons, a grid for accelerating electrons and an annular anode. The voltage difference between cathode and anode must be very high, usually between several thousand volts and three million volts. It can emit and form an electron beam with uniform speed, so the stability of accelerating voltage is required to be not less than one ten thousandth. ③ Sample rack: the sample can be stably placed on the sample rack. In addition, there are often devices that can be used to change samples (such as moving, rotating, heating, cooling, stretching, etc.). (4) detector: used to collect electronic signals or secondary signals.

1.2 Basic principles Different types of electron microscopes have different imaging principles, but they all use electromagnetic fields to deflect and focus electron beams, and then study the structure of matter according to the principle of interaction between electrons and matter. Among them, the electron beam generated by the transmission electron microscope is converged by the condenser and evenly irradiated to the area to be observed on the sample, and the incident electrons interact with the sample substance. Because the sample is very thin, most electrons penetrate the sample, and its intensity distribution corresponds to the morphology, microstructure and structure of the observed sample area. The electrons projected from the sample are amplified by the three-stage magnetic lens and projected onto the phosphor screen for observing the pattern, and the phosphor screen converts the electron intensity distribution into the light intensity distribution visible to human eyes, thereby displaying an image corresponding to the shape, organization and structure of the sample on the phosphor screen. Scanning electron microscope (SEM) uses focused electron beam driven by coil to scan and image the sample surface point by point, and the imaging signal is secondary electron, backscattered electron or absorbed electron. The secondary electrical signal is collected by the detector and converted into electrical signal, and the secondary electronic image of the surface morphology of the reaction sample is obtained after processing. Backscattered electron imaging reflects the element distribution of the sample and the outline of different phase composition regions. In addition, due to the short de Broglie wavelength of electrons, the resolution is much higher than that of optical microscope, which can reach 0. 1 ~ 0.2 nm, and the magnification is from tens of thousands to millions.

1.3 technical development history The first transmission electron microscope (TEM) in the world was successfully developed by German scientists ruska and Noel in 193 1 year. After World War II, ruska continued to study and improve TEM, and made a microscope with a magnification of more than 654.38+ 10,000 times, thus winning the Nobel Prize in Physics. On the basis of TEM, British engineer Charles invented the world's first scanning electron microscope (SEM) in 1952. Scanning electron microscope is mainly used to observe thick samples with height difference and roughness, so it highlights the depth of field effect in design, and is generally used to analyze fractures and natural surfaces without manual treatment. However, transmission electron microscope (TEM) highlights high resolution. High-resolution ultrastructural images can be obtained by observing samples with TEM, which is widely used in materials science and biology, and is also a diagnostic tool for pathology. The key of this technology is the preparation of ultra-thin slices. After that, field emission scanning electron microscope (FE-SEM), field ion microscope (FIM), low energy electron diffraction (LEED), Auger spectrometer (AES) and photoelectron spectrometer (ESCA) were born one after another, which played an important role in the research of various scientific fields. 198 1 year, G. Binnig and H. Rohrer successfully developed the world's first scanning tunneling microscope (STM) and won the Nobel Prize in Physics. Its appearance enables human beings to observe the arrangement of single atoms on the surface of materials and the physical and chemical properties related to the surface electronic behavior for the first time in real time, which is recognized by the international scientific community as one of the top ten scientific and technological achievements in the world in the 1980s. Scanning tunneling microscope (STM) is a new instrument, which uses the tunneling current between the conductor tip and the sample, and uses a precise piezoelectric crystal to control the conductor tip to scan along the surface of the sample, thus recording the surface morphology of the sample on the atomic scale. Its resolution reaches 1 nm ~ 2 nm, and it can be used to study the surface morphology of various metals, semiconductors and biological samples, as well as surface deposition, surface atomic diffusion, nucleation and growth, adsorption and desorption of surface particles. After the appearance of STM, a series of new microscopic techniques with similar working principles have been developed, including atomic force microscope (AFM) and transverse force microscope (LFM). These microscopes that scan and image samples based on probes are collectively called scanning probe microscopes (SPM). Scanning probe microscope is the most important and basic method in nano-metrology, nano-characterization and measurement. It can use the atomic probe and the surface of the sample to be measured as the main components, complete the scanning between the probe and the sample in X and Y directions, and simulate the fluctuation of the sample surface in Z direction. The physical quantity produced by the interaction between the probe and the sample changes with the fluctuation of the sample surface to observe the surface morphology of the sample. The instrument has high resolution, the horizontal resolution can reach 0. 1nm, and the vertical resolution can reach 0.0 1nm. It can directly observe and determine the three-dimensional image of the sample, and can be observed in the atmosphere, vacuum or even high or low temperature liquid. It can be detected without touching the sample, so it will not damage the sample and does not need electron beam irradiation, so it will not cause radiation damage to the sample.

Two. Development of Electron Microscope Technology in China 1958, China successfully developed the first electron microscope; 1988, Bai Chunli and Yao Junen of China Academy of Sciences developed the first STM in China. [2] In 2000, China Electron Microscope Society counted less than 2,000 sets of Chinese mainland. After China's accession to the WTO, economic development, scientific research, education and industrial structure are all upgrading. At present, the electron microscope market in China is increasing by nearly 100 sets every year. It can be predicted that the market capacity of electron microscope in China will rank first in the world in the next few years. For the electron microscope in China market, the market share of Japanese electronics exceeds 50%, ranking first. Followed by Fei (formerly Philips Electron Microscopy Department), Hitachi (agent), Germany (formerly Leo) and Shimadzu, Japan. Domestic manufacturers are mainly Zhongke Yike, Nanjing Jiangnan Optoelectronics and Shanghai Institute of Electronic Optics Technology, and their products are mainly concentrated in the low-end scanning electron microscope market. As far as the overall market situation is concerned, the domestic market share of domestic electron microscope is less than 10%. It can be seen that there is still a lot of room for improvement in China's domestic electron microscope. In terms of types, scanning electron microscope accounts for 63.6 1% of the total number of electron microscopes in China, and transmission electron microscope accounts for 36.39%, which shows that scanning electron microscope has a wider user base in China. [3]

Third, the future development trend of electron microscope technology

3. 1 Remote Electron Microscope Technology Since the 1990s, with the development of computer technology and network technology, remote electron microscopes have gradually appeared, which can display the real-time information obtained in the laboratory to remote users, so that they can watch the sample images in real time through the Internet and remotely operate the instruments to complete the experiments. [4] The key of remote electron microscope technology lies in image acquisition, compression and transmission. In the aspect of image acquisition, the present electron microscope has made great progress. The old electron microscope mostly uses digital camera and video capture card to collect images, and the new electron microscope mostly uses VGA capture card to collect images, which has become the future development trend. In addition, new methods of using software to collect images are gradually emerging. In the early days, JPEG image compression was used for image compression, that is, remote users saw a series of independent static sample images. Now, with the development of technology, MPEG4-4, H.264 and other video compression algorithms are gradually applied to the compression of sample images. At present, the transmission of sample images is mainly through TCP and UDP protocols, but these two protocols occupy too much bandwidth and the transmission effect is not ideal. In order to improve the transmission performance, the "pyramid" network transmission model and proprietary transmission network of special data transmission system are being studied, which is also the improvement direction of remote electron microscope at this stage. 1990, Karl Zmola and others realized the network transmission of scanning electron microscope sample images, and established the real-time transmission system of remote electron microscope sample images for the first time. Subsequently, American universities have established their own SEM remote systems. The efficiency of sample transmission has also made great progress. Initially, in the 800Mb optical fiber network, the transmission efficiency of sample images was every 17 seconds 1 frame. By 2000, in the network of 1~2Mb, the transmission of sample images can reach 5 frames per second. There is still a lot of room for improvement in technology. In China, although there are thousands of electron microscopes in universities and scientific research institutions, they still can't meet the growing application demand. Therefore, the research of remote electron microscope technology has important application value to our country.

3.2 Low-temperature electron microscope technology Low-temperature electron microscope technology is a technology that uses freezing (physical) method to prepare biological samples and observe them, so it is widely used in biological histology. Compared with conventional electron microscope technology (chemical method), it can keep the physiological state of the sample to the maximum extent, and can be used to study the dynamic process of biological macromolecules and analyze the three-dimensional structure of nuclear tissue.

3.3 Three-dimensional reconstruction technology under low-temperature electron microscope The three-dimensional imaging technology of electron microscope is the perfect combination of electron microscope and computer. It uses an electron microscope to collect the two-dimensional projection image of the sample, and reconstructs the three-dimensional spatial structure of the sample through computer processing. Three-dimensional imaging technology is widely used in the field of biology, especially the three-dimensional structure analysis of protein. Early three-dimensional imaging technology mainly used heavy metal salt solution to dye samples.