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Exploring the micro world:from optical microscope to electron microscope

Human’s naked eye resolution is about 0.1mm. How do we see bacteria, viruses and even protein structures step by step? This is inseparable from this group of”obsessive-compulsive disorder”.

Interview experts:Zhangdetian (Professor, national biomedical analysis center, Academy of Military Medical Sciences)

“I was very surprised to see many tiny living microorganisms in the water. They are so beautiful and moving. Some of them are like spears passing through the water, some are like gyros spinning in place, and others are nimbly wandering and moving forward in groups. You can imagine them as a group of flying mosquitoes.”

In 1675, a small civil servant of Delft City Hall in the Netherlands wrote such a letter to the Royal Society of England, describing to the members of the society the wonderful scene he observed with his own microscope. As an academic discussion letter sent to the most famous academic organization in Europe at that time, the civil servant did not carry out a large and rigorous but boring scientific demonstration, but left the childlike surprise and joy of discovering new things between the lines in plain language.

This little civil servant who was unknown at that time was Anthony van Leeuwenhoek, the famous pioneer of Microbiology and microscopy. In the past 50 years, Leeuwenhoek has observed micro organisms such as bacteria, muscle fibers and sperm cells with his microscope, and has sent more than 300 letters to the British royal society to discuss his new findings. It is with the unremitting insistence of Leeuwenhoek that human eyes for observing the world have finally come to the microbial level.


First generation microscope:Dispel the fog of microbial world

Leeuwenhoek can discover the colorful microbial world, mainly thanks to his talent in lens making. In his life, he produced more than 400 microscopes, which is very different from the microscopes we are familiar with today. Most of lewenhoek’s microscopes belong to single lens microscopes, which are only composed of a small brass plate. When using, he needs to lean back and face the copper plate to the sun for observation. Leeuwenhoek quickly became a”net celebrity” in the scientific community at that time by virtue of his series of amazing discoveries.

However, it was Robert hooker, a British scientist of the same period, who really laid the theoretical foundation of microscopy. In 1665, when Leeuwenhoek was still studying lens making techniques, Hooke, who was responsible for scientific experiments at the British royal society, made a microscope. Unlike the single lens microscope used by Leeuwenhoek, this is a compound microscope, and its working principle and shape are very close to modern optical microscopes.

Hooker observed a piece of cork with this microscope and found a dense lattice structure, which was similar to the single room where monks lived at that time. Therefore, Hooker named this structure with the word”cell” in English, which is translated as”cell” in modern times. Before long, Hooker wrote a book, micrograph, which included this important observation.

Hooke’s research results soon attracted Leeuwenhoek’s attention. He once studied Hooke’s microscope, but finally used a self-made single lens microscope to observe. The reason lies in the serious color difference of Hooke microscope. The so-called color difference means that when the light passes through the lens, the light of different colors will focus on different points due to different refractive index, so that the image of the sample is surrounded by a layer of color spots, which seriously affects the definition.

The solution proposed by Leeuwenhoek is also very simple. It is to work hard on the fineness of lens grinding, make a single lens into small glass beads, and embed them into the fine holes of the brass plate, so as to avoid the interference of color difference to the imaging to the greatest extent on the basis that the magnification is not lower than that of Hooke’s microscope. But the cost is that it is necessary to face the sun when observing, which is very harmful to the observer’s eyes.

In addition to chromatic aberration, early microscopes also had the problem of spherical aberration, that is, when the light was refracted through the lens, the light near the center and the edge could not concentrate the image at one point, making the image blurred. Since the birth of microscope, chromatic aberration and spherical aberration have become”inherent diseases”, which have restricted people’s pace of marching into the micro world. It was not until the 19th century that optical microscopy completed a substantial transformation with the help of the industrial revolution, thus fundamentally solving these two problems.


Challenge chromatic aberration and spherical aberration:Gradually clear micro perspective

Firstly, in 1830, a British Amateur microscopist named Liszt first challenged the spherical aberration. He creatively used several lens groups with specific spacing to successfully reduce the influence of spherical aberration.

After that, the main position for improving the microscope was quickly transferred to Germany. Zeiss optical factory, established in 1846, became the leader in the next century. In 1857, Zeiss factory developed the first modern duplex microscope and successfully entered the market. However, during the development and production process, Zeiss was also suffering from color difference:the common practice of increasing the number of lenses at that time could improve the magnification of the microscope, but still could not eliminate the interference of color difference on the imaging clarity.

In 1872, Professor Ernst Abel of Jena University in Germany put forward a perfect microscope theory, which explained in detail the imaging principle, numerical aperture and other scientific problems of optical microscope. Zeiss also quickly invited Professor Abbe to join us and developed a number of epoch-making optical components, including apochromatic lenses, which eliminated the influence of chromatic aberration in one fell swoop.

With the technical support of Professor Abbe, the microscope of Zeiss factory has become a leader in similar products, and soon became a hot commodity in major laboratories in Europe and America, and laid the basic form of modern optical microscope. Before long, Zeiss brought in the famous chemist Otto Schott to apply the lithium glass developed by Zeiss with brand-new optical properties to its own products. In 1884, Zeiss, together with Abbe and Schott, established the”Jena glass factory” to produce professional lenses for microscopes.

With the rapid development of microscope technology, various modern biological theories have been continuously improved. Through the high-resolution lens, various complex structures in the micro world are gradually presented to human beings in a concrete form.

Since most biological structures at the micro level are colorless and transparent, in order to make them visible under the lens, scientists at that time generally dyed biological samples to improve the contrast and facilitate observation. The biggest limitation of this method is that the toxicity of the dye itself often destroys the organizational structure of microorganisms. In this period, the backward materials of the dye could not dye some specific tissues.

It was not until 1935 that the Dutch scholar zenik discovered the phase contrast principle and attributed its success to the microscope. This phase contrast microscopy technology uses the extremely small phase difference generated by light passing through transparent objects to image, so that the microscope can clearly observe colorless and transparent biological samples. Zenik himself won the 1953 Nobel Prize in physics for his discovery.

Professor, national biomedical analysis center, Academy of Military Medical Sciences, Zhangdetian, who has long been committed to the research in the field of electron microscope, told reporters:”the resolution of human eyes is about 0.1mm, while the resolution of optical microscope can reach the level of 0.2 microns (1mm =1000 microns), and bacteria and cells can be seen. However, due to the fluctuation of light, diffraction phenomenon limits the further improvement of the resolution of optical microscope.”

After the end of World War II, with the continuous application of various new theories and technologies, optical microscope has made great progress, but also during this period, the potential of optical microscope has been explored to the limit. Professor Abbe, who has made great contributions to Zeiss factory and even the whole microscopy, put forward the”resolution limit theory”, which holds that the resolution limit of ordinary optical microscopes is 0.2 microns, and no small object can do anything – this theory is also known as”Abbe limit”, which is like a barrier blocking the exploration vision of human beings in front of the door of the deeper micro world, forcing scientists to find another way.


Electron microscope:Find another way and rediscover

Since there is such a short board in visible light, can we use other short wavelength beams to achieve a breakthrough in resolution? Zhang Detian further said:”after 1924, people found a medium electron with shorter wavelength in the field of matter, and invented the electron microscope. Its resolution reached the level of 0.1 nm.”

In 1931, the German scientist Knorr and his student Ruska installed a discharge electron source and three electron lenses on a high-voltage oscilloscope to make the world’s first electron microscope, which opened up a new way for human beings to explore the micro world.

The electron microscope is completely free from the shackles of Abbe limit, and its resolution is far beyond that of the optical microscope at that time. Rusca improved the electron microscope the following year, and the resolution reached the nanometer level (1 micron =1000 nm). At this depth of observation, human beings have finally witnessed a microorganism – A virus – smaller than bacteria. In 1938, Ruska saw the real body of tobacco mosaic virus with an electron microscope. At this time, 40 years had passed since the virus was confirmed to exist.

Regarding the invention of the electron microscope technology, Zhang Detian commented:”the electron microscope is the key and tool for people to understand the ultra micro world. It solves the problem that the optical microscope is limited by the wavelength of natural light, and improves people’s understanding of the world from the cellular level to the molecular level.” From the millimeter scale that can only be observed by the naked eye, to the micron scale that can be reached by the optical microscope, and then to the nano scale that can be further explored by the electron microscope, micro imaging technology is rapidly breaking through the cognitive limit of human beings on the micro world.

However, the shortcomings of the electron microscope itself are becoming more and more obvious. Because electron acceleration can only be realized under vacuum, biological samples are often dehydrated and dried under vacuum environment, which means that the biological samples in the living state can not be observed by the electron microscope at all. In addition, the electron beam itself is easy to destroy the biological molecular structure on the surface of the sample, which leads to the loss of many key information of the sample itself. This stubborn disease has puzzled scientists for many years since then.

Until 1981, binrich and lorel, two researchers of IBM Zurich laboratory, first solved the problem of electron beam damaging the sample structure by using a method that seemed quite”unorthodox” at that time. They used the”tunneling effect” in quantum physics to make a scanning tunneling microscope.

Unlike traditional optical and electronic microscopes, this kind of microscope has no lens. When working, a probe is used to approach the sample and a voltage is applied between them. When the probe is only nanometer away from the sample, a tunneling effect will occur – electrons pass through the tiny gap to form a weak current. This current will change with the change of the distance between the probe and the sample. People can indirectly obtain the approximate shape of the sample by measuring the change of the current. Because there is no electron beam in the whole process, scanning tunneling microscope fundamentally avoids the damage of accelerated electrons to the surface of biological samples.

Scanning tunneling microscope (STM) is also called”atomic force microscope” today观察生物样品表面形貌结构的变化规律,原子力显微镜是有其独特优势的”,张德添向记者解释说,“如果条件允许,还可以检测生物大分子间相互作用力的大小,为结构与功能关系研究提供便利。”

1986年,宾尼希和罗雷尔凭借扫描隧道显微镜,获得当年的诺贝尔物理学奖,有趣的是,与他们一起分享荣誉的,还有当初发明电子显微镜的鲁斯卡,当时的他已是耄耋老人,而他的恩师克诺尔也早已作古。新老两代电子显微镜技术的里程碑人物同台领奖,成为当时物理学界的一段佳话。


老树新芽:突破“阿贝极限”的光学显微镜

电子显微镜在问世之后的几十年间,极大拓展了人类对生物、化学、材料和物理等领域认知疆界。而无论是鲁斯卡,还是宾尼希和罗雷尔,他们所作的贡献不仅让自己享誉世界,还助力其他领域的学者登上荣誉之巅。

比如英国化学家艾伦·克鲁格凭借对核酸与蛋白复杂体系的研究获得1982年度诺贝尔化学奖,而他的科研成果正式依靠高分辨电子显微镜技术和X光衍射分析技术而取得的。在克鲁格获奖的当年,以色列化学家达尼埃尔·谢赫特曼更是使用一台电子显微镜,发现了准晶体的存在,并独享了2011年的诺贝尔化学奖。

目前,电子显微镜已经成为金属、半导体和超导体领域研究的主力军。但在生物和医学领域,电子显微镜本身对生物样品的损害,依旧是难以逾越的技术难题。于是不少科学家开始从两条路径上寻求解决之道:

一条是研发冷冻电镜技术,这种技术并不改变电子显微镜整体的工作模式,而是从生物样品本身入手,对其进行超低温冷冻处理。这样状态下,即使处在真空环境中,样品也能保持原有的形态特征与生物活性。“由于观测温度低,生物样品也处于含水状态,分子也处于天然状态,样品对辐射的耐受能力得以提高。我们可以将样品冻结在不同状态,观测分子结构的变化。”张德添向记者解释道。

瑞士物理学家雅克·杜波切特、美国生物学家乔基姆·弗兰克和英国生物学家理查德·亨德森凭借这项技术分享了2017年度诺贝尔化学奖。新冠疫情暴发后,冷冻电镜技术又为人类研究和抗击疫情做出了突出贡献。2020年,西湖大学周强实验室就利用这种技术,首次成功解析了此次新冠病毒的受体—ACE2的全长结构,让人类对新冠病毒的认识向前迈出了关键性一步。

另一条路径是从传统的光学显微镜入手。在电子显微镜的黄金时代,不少科学家就开始着手研制超高分辨率光学显微镜,甚至开始尝试突破一直以来困扰光学显微镜的“阿贝极限”,而“荧光技术”就成为实现这一切的关键。

早在19世纪中叶,科学家们就发现:某些物质在吸收波长较短而能量较高的光线(比如紫外光)时,能将光源转化为波长较长的可见光。这种现象后来被定义为“荧光现象”。

荧光现象在自然界是普遍存在的,这一现象背后的原理也在20世纪迅速被应用在光学显微镜上。1911年,德国科学家首次研制出荧光显微镜装置,用荧光色素对样品进行荧光染色处理,并以紫外光激发样品的荧光物质发光,但成像效果不佳,而且把荧光物质当作染色剂,和早期的染色剂一样,本身的毒性会伤害活体样品。

直到1974年,日本科学家下村修发现了绿色荧光蛋白,其毒性远弱于以往的荧光物质,是对活体标本进行荧光标记的理想材料——这一发现成为日后科学家突破“阿贝极限”的有力武器。

时间来到1989年,供职于美国IBM研究中心的科学家莫尔纳首次进行了单分子荧光检测,使得光学显微镜的检测尺度精确到纳米量级成为可能。随后在莫尔纳的基础上,美国科学家贝齐格开发出一套新的显微成像方法:控制样品内的荧光分子,让少量分子发光,借此确定分子中心和每个分子的位置,通过多次观察呈现出纳米尺度的图像。通过这种方法,贝齐格轻而易举地突破了光学显微镜的阿贝极限。

几乎在同时,德国科学家斯特凡·赫尔在一次光学研究中突发奇想:根据荧光现象原理,如果用镭射光激发样品内的荧光物质发光,同时用另一束镭射光消除样品体内较大物体的荧光,这样就只剩下纳米尺度的分子发射荧光并被探测到,不就能在理论上得到分辨率大于0.2微米的微观成像了吗?

他随即开始了试验,并制成了一台全新显微镜,将光学显微镜分辨率下探到了0.1微米的水平。困扰光学显微技术百年的阿贝极限难题,就这样历经几代科学家的呕心沥血,终于在本世纪初被成功攻克。莫尔纳、贝齐格和赫尔三位科学家更是凭借“超分辨率荧光显微技术”分享了2014年度的诺贝尔化学奖。

时至今日,在探索微观世界的征途上,光学显微镜和电子显微镜互有长短、相得益彰。当然在实际应用中,科学家越来越依赖于将多种显微成像技术结合使用。比如今年5月,英国弗朗西斯·克里克研究所就依托钙化成像技术、体积电子显微技术等多种显微成像技术,成功获得了人类大脑神经网络亚细胞图谱。在未来,多种显微成像技术相结合,各施所长,将进一步完善我们在生物、医学、化学和材料等领域的知识结构,把这个包罗万象的奇妙世界更完整地呈现在我们眼前。