Humans can see these "little guys" only because of this group of obsessive compulsive disorder

The human eye can only distinguish about 0.1 mm. How do we see bacteria, viruses, and even protein structures step by step? This is inseparable from this group of "obsessive-compulsive disorder".

Written by reporter Duan Ran Edited by Liu Zhao

New Media Editor/Li Yunfeng

Interview experts

Zhang Detian (Professor at the National Center for Biomedical Analysis, Academy of Military Medical Sciences)

"I was very surprised to see so many tiny living microorganisms in the water. They were so beautiful and moving. Some of them pierced the water like spears, some spun in place like spinning tops, and some moved nimbly and in groups. You can imagine them as a group of flying mosquitoes."

In 1675, a small civil servant from the Delft City Hall in the Netherlands wrote a letter to the Royal Society of London, describing to the members of the society the wonderful sights he had observed with a homemade microscope. As an academic discussion letter sent to the most prestigious academic organization in Europe at the time, the civil servant did not conduct a long, rigorous but boring scientific argument, but used plain language to leave behind the childlike surprise and joy of discovering new things between the lines.

This little-known civil servant was the famous pioneer of microbiology and microscopy, Antonie van Leeuwenhoek. Over a 50-year period, Leeuwenhoek used his microscope to observe microscopic organisms such as bacteria, muscle fibers and sperm cells, and sent more than 300 letters to the Royal Society of London to discuss his new discoveries. It was with Leeuwenhoek's unremitting persistence that humans finally observed the world at the microbial level.

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The first microscope: Uncovering the mystery of the microbial world

Leeuwenhoek was able to discover the colorful world of microorganisms mainly due to his talent in lens making. He made more than 400 microscopes in his lifetime. They were very different from the microscopes we know today. Most of Leeuwenhoek's microscopes were single-lens microscopes, consisting of only a small brass plate, which required lying on one's back with the plate facing the sun for observation. Leeuwenhoek quickly became an "Internet celebrity" in the scientific community at the time with his series of amazing discoveries.

▲Image of a microscope designed by Leeuwenhoek in the 17th century (Photo source: Visual China)

However, it was Robert Hooke, a British scientist of the same period, who laid the theoretical foundation of microscopy. In 1665, when Leeuwenhoek was still studying the lens making technique, Hooke, who was in charge of scientific experiments at the Royal Society of England, made a microscope. Unlike the single-lens microscope used by Leeuwenhoek, this was a compound microscope, and its working principle and appearance were very close to modern optical microscopes.

Hooke used this microscope to observe a piece of cork and found a dense lattice structure, which was very similar to the single room where monks lived at that time. Therefore, Hooke named this structure after the English word for single room, "cell", which is translated as "cell" in modern times. Soon after, Hooke wrote the book "Microscopic Atlas", in which he recorded this important observation.

Hooke's research results soon attracted the attention of Leeuwenhoek. He once studied Hooke's microscope, but finally used a homemade single-lens microscope for observation. The reason was that Hooke's microscope had serious chromatic aberration problems. Chromatic aberration means that when light passes through a lens, different colors of light will focus on different points due to different refractive indices, causing the image of the sample to be surrounded by a layer of color spots, seriously affecting the clarity.

Leeuwenhoek's solution was also very simple, which was to work hard on the fineness of lens grinding, make single lenses into small glass beads, and embed them into the pores of brass plates. In this way, the interference of chromatic aberration on imaging can be avoided to the greatest extent on the basis of the magnification not lower than that of Hooke's microscope. But the cost was that since observation needed to be made under the sun, it was very harmful to the observer's eyes.

In addition to chromatic aberration, early microscopes also had spherical aberration problems, that is, when light is refracted through the lens, the light near the center and the light near the edge cannot focus the image on one point, making the image blurry. Since the birth of the microscope, chromatic aberration and spherical aberration have become "innate diseases" that have always restricted people's pace of advancing into the microscopic world. It was not until the 19th century that optical microscopy technology completed a substantial transformation with the help of the Industrial Revolution, thus fundamentally solving these two problems.

▲Using a single lens to magnify the image of an object (Image source/Technology Networks)

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Challenging Chromatic and Spherical Aberrations: A Gradually Clearer Microscopic Perspective

First, in 1830, a British amateur microscopist named Lister first challenged spherical aberration. He creatively used several lens groups with specific spacing to successfully reduce the impact of spherical aberration.

After that, the main battlefield for improving microscopes quickly shifted to Germany, where the Zeiss Optical Factory, founded in 1846, became the leader in the next century. In 1857, the Zeiss factory developed the first modern compound microscope and successfully entered the market. However, during the development and production process, Zeiss also suffered from chromatic aberration: the common practice of increasing the number of lenses at the time, although it could increase the magnification of the microscope, still could not eliminate the interference of chromatic aberration on image clarity.

In 1872, Professor Ernst Abbe of the University of Jena in Germany proposed a complete theory of microscopy, detailing the imaging principle of optical microscopes, numerical aperture and other scientific issues. Zeiss also quickly invited Professor Abbe to join 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 microscopes produced by the Zeiss factory became the best among similar products, and soon became popular in major laboratories in Europe and the United States, laying the foundation for the basic form of modern optical microscopes. Soon, Zeiss brought in the famous chemist Otto Schott to join the company and applied the lithium glass with new optical properties developed by him to its own products. In 1884, Zeiss joined forces with Abbe and Schott to establish the "Jena Glass Factory" to produce professional lenses for microscopes.

▲Compound microscope produced in 1862 (Photo/Manfred Stich)

The rapid development of microscope technology has also enabled various modern biological theories to be continuously improved. Through high-resolution lenses, various complex structures in the microscopic world are gradually presented to human eyes in a concrete form.

Since most biological structures at the microscopic level are colorless and transparent, in order to make them clearly visible under the camera, scientists at the time generally dyed biological samples to increase contrast and facilitate observation. The biggest limitation of this method is that the toxicity of the dye itself often destroys the tissue structure of microorganisms. The backward materials of the dyes at this time also made it impossible to dye certain specific tissues.

It was not until 1935 that Dutch scholar Zernike discovered the phase contrast principle and successfully applied it to microscopes. This phase contrast microscopy technique uses the extremely subtle phase difference produced by light passing through transparent objects to form an image, allowing the microscope to clearly observe colorless and transparent biological samples. Zernike himself won the 1953 Nobel Prize in Physics for this discovery.

Zhang Detian, a professor at the National Center for Biomedical Analysis of the Academy of Military Medical Sciences, who has long been committed to the research of electron microscopy, told reporters: "The human eye can only resolve about 0.1 millimeters, while the optical microscope can resolve up to 0.2 microns (1 millimeter = 1000 microns), which can see bacteria and cells. However, due to the wave nature of light, the diffraction phenomenon limits the further improvement of the resolution of optical microscopes."

After the end of World War II, with the continuous application of various new theories and technologies, optical microscopes have made great progress, but it was also during this period that the potential of optical microscopes had been explored to the limit. Professor Abbe, who made great contributions to the Zeiss factory and even the entire microscopy, proposed the "resolution limit theory", arguing that the resolution limit of ordinary optical microscopes is 0.2 microns, and objects smaller than that are powerless - this theory is also called the "Abbe limit", which is like a barrier blocking human exploration before the door to a deeper microscopic world, forcing scientists to find another way.

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Electron microscopy: a new approach to discovery

Since visible light has such a shortcoming, can we use other light beams with shorter wavelengths to achieve a breakthrough in resolution? Zhang Detian further introduced: "After 1924, people found a medium with a shorter wavelength in the material field - electrons, and invented the electron microscope, whose resolution ability reached the level of 0.1 nanometers."

In 1931, 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, opening up a new way of thinking for human exploration of the microscopic world.

▲The transmission electron microscope developed by Ruska in 1933 (Photo source/Wikimedia Commons)

The electron microscope was completely free from the shackles of the Abbe limit, and its resolution far exceeded that of the optical microscope at that time. Ruska improved the electron microscope the following year, and the resolution reached the nanometer level (1 micrometer = 1000 nanometers). At this observation depth, humans finally saw microorganisms smaller than bacteria - viruses. In 1938, Ruska used an electron microscope to see the true form of the tobacco mosaic virus, and 40 years had passed since the virus was confirmed to exist.

▲Tobacco mosaic virus under a transmission electron microscope (Photo source/The Nobel Prize)

Regarding the invention of electron microscopy technology, Zhang Detian commented: "The electron microscope is the key and tool for people to understand the ultra-microscopic world. It solves the problem that optical microscopes are limited by the wavelength of natural light, and raises 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 micrometer scale that can be reached by optical microscopes, and then to the nanometer scale that electron microscopes can further explore, microscopic imaging technology is rapidly breaking through the limits of human cognition of the microscopic world.

However, the shortcomings of the electron microscope itself became increasingly obvious. Since electron acceleration can only be achieved under vacuum conditions, biological samples often have to be dehydrated and dried under vacuum conditions, which means that the electron microscope cannot observe biological samples in a living state at all. In addition, the electron beam itself can easily destroy the biological molecular structure on the surface of the sample, which causes the sample itself to lose a lot of key information. This stubborn problem has troubled scientists for many years.

It was not until 1981 that two researchers at IBM's Zurich laboratory, Bennig and Rohrer, first solved the problem of electron beam damage to sample structure using a method that seemed quite "heretical" at the time. They used the "tunnel effect" in quantum physics to create a scanning tunneling microscope.

Unlike traditional optical and electron microscopes, this microscope does not even have a lens. When working, a probe is used to approach the sample and a voltage is applied between the two. When the probe is only nanometers away from the sample, a tunneling effect occurs - electrons pass through this tiny gap, forming a weak current. This current changes with the distance between the probe and the sample. By measuring the change in current, people can indirectly obtain the approximate shape of the sample. Since there is no electron beam involved in the whole process, the scanning tunneling microscope fundamentally avoids the damage of accelerated electrons to the surface of biological samples.

Scanning tunneling microscopes are also called "atomic force microscopes" today. "At the micrometer or even nanometer level, atomic force microscopes have unique advantages in dynamically observing the changes in the surface morphology and structure of biological samples," Zhang Detian explained to reporters. "If conditions permit, it can also detect the magnitude of the interaction force between biological macromolecules, providing convenience for the study of the relationship between structure and function."

In 1986, Binnig and Rohrer won the Nobel Prize in Physics for their scanning tunneling microscope. Interestingly, they shared the honor with Ruska, who invented the electron microscope. At that time, he was already an octogenarian, and his mentor Knorr had long passed away. The two generations of milestone figures in electron microscope technology, the old and the new, received the award on the same stage, which became a good story in the physics community at that time.

▲In 1981, Bennig and Rohrer developed the scanning tunneling microscope. Five years later, they won the Nobel Prize in Physics (Image source/The Nobel Prize)

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New buds from an old tree: An optical microscope that breaks the "Abbe limit"

In the decades since its invention, the electron microscope has greatly expanded the boundaries of human cognition in biology, chemistry, materials, physics and other fields. Whether it is Ruska, Binnig or Rohrer, their contributions have not only made themselves world-renowned, but also helped scholars in other fields to reach the pinnacle of honor.

For example, British chemist Alan Krug won the 1982 Nobel Prize in Chemistry for his research on complex systems of nucleic acids and proteins, and his scientific research results were achieved by relying on high-resolution electron microscopy technology and X-ray diffraction analysis technology. In the same year that Krug won the award, Israeli chemist Daniel Shechtman used an electron microscope to discover the existence of quasicrystals and won the 2011 Nobel Prize in Chemistry alone.

At present, electron microscopes have become the main force in the research of metals, semiconductors and superconductors. However, in the fields of biology and medicine, the damage caused by electron microscopes to biological samples is still an insurmountable technical problem. Therefore, many scientists have begun to seek solutions from two paths:

One is to develop cryo-electron microscopy technology, which does not change the overall working mode of the electron microscope, but starts with the biological sample itself and performs ultra-low temperature freezing treatment on it. In this state, even in a vacuum environment, the sample can maintain its original morphological characteristics and biological activity. "Due to the low observation temperature, the biological sample is also in a hydrated state, and the molecules are also in a natural state, and the sample's tolerance to radiation is improved. We can freeze the sample in different states and observe changes in the molecular structure." Zhang Detian explained to reporters.

Swiss physicist Jacques Dubochet, American biologist Joachim Frank and British biologist Richard Henderson shared the 2017 Nobel Prize in Chemistry for this technology. After the outbreak of the COVID-19 pandemic, cryo-electron microscopy technology has made outstanding contributions to human research and the fight against the pandemic. In 2020, Zhou Qiang's laboratory at Westlake University used this technology to successfully analyze the full-length structure of ACE2, the receptor of the new coronavirus, for the first time, taking a crucial step forward in human understanding of the new coronavirus.

▲Cryo-EM density map of ACE2-B0AT1 complex (Image source/biorxiv)

Another approach is to start with traditional optical microscopes. In the golden age of electron microscopes, many scientists began to develop ultra-high-resolution optical microscopes, and even began to try to break through the "Abbe limit" that has always plagued optical microscopes, and "fluorescence technology" became the key to achieving all this.

As early as the mid-19th century, scientists discovered that when certain substances absorb shorter wavelength and higher energy light (such as ultraviolet light), they can convert the light source into longer wavelength visible light. This phenomenon was later defined as "fluorescence".

Fluorescence is ubiquitous in nature, and the principle behind it was quickly applied to optical microscopes in the 20th century. In 1911, German scientists developed the first fluorescence microscope, which used fluorescent pigments to dye samples and used ultraviolet light to stimulate the fluorescent substances in the samples to emit light. However, the imaging effect was poor, and the fluorescent substances were used as dyes, which, like early dyes, were toxic and could harm living samples.

It was not until 1974 that Japanese scientist Osamu Shimomura discovered green fluorescent protein, which is far less toxic than previous fluorescent substances and is an ideal material for fluorescent labeling of living specimens. This discovery became a powerful weapon for scientists to break through the "Abbe limit" in the future.

In 1989, Molnar, a scientist working at the IBM Research Center in the United States, conducted the first single-molecule fluorescence detection, making it possible for optical microscopes to detect objects accurately at the nanometer level. Later, based on Molnar's work, American scientist Betzig developed a new microscopic imaging method: controlling the fluorescent molecules in the sample, allowing a small number of molecules to emit light, thereby determining the molecular center and the position of each molecule, and presenting nanometer-scale images through multiple observations. With this method, Betzig easily broke through the Abbe limit of optical microscopy.

▲Fibroblasts, one of the most common cells in mammalian connective tissue, are shown by the prototype of photoactivated localization microscopy (PALM). The DNA in the nucleus (blue), mitochondria (green) and cytoskeleton (red) is clearly visible (Image source/NIH)

Almost at the same time, German scientist Stefan Hell had a sudden idea during an optical research: According to the principle of fluorescence phenomenon, if laser light is used to excite the fluorescent substance in the sample to emit light, and another beam of laser light is used to eliminate the fluorescence of larger objects in the sample, then only the nanoscale molecules will emit fluorescence and be detected. In theory, can't we get microscopic imaging with a resolution greater than 0.2 microns?

He immediately began to experiment and built a new microscope, lowering the resolution of optical microscopes to 0.1 microns. The Abbe limit problem that had plagued optical microscopy technology for a century was finally solved at the beginning of this century after the painstaking efforts of several generations of scientists. Molnar, Betzig and Hull shared the 2014 Nobel Prize in Chemistry for their "super-resolution fluorescence microscopy technology."

Today, in the journey of exploring the microscopic world, optical microscopes and electron microscopes have their own strengths and weaknesses and complement each other. Of course, in practical applications, scientists are increasingly relying on the combination of multiple microscopic imaging technologies. For example, in May this year, the Francis Crick Institute in the UK successfully obtained a subcellular map of the human brain neural network by relying on calcification imaging technology, volume electron microscopy technology and other microscopic imaging technologies. In the future, the combination of multiple microscopic imaging technologies, each with its own strengths, will further improve our knowledge structure in the fields of biology, medicine, chemistry and materials, and present this all-encompassing wonderful world more completely before our eyes. ■