" There is no function without anatomy ". Camillo Golgi, Nobel Prize in Medicine in 1906, wrote this at the end of the XNUMXth century about his studies on central and peripheral nerve cells. In other words, the function reshapes the shape of cells, and therefore microscopic observations become essential to understand its physiological mechanisms.
It is no coincidence that the first person who understood that nerves were made up of a set of cables and did not represent a kind of channel with a soft substance inside, as in the case of blood vessels, was Anton Van Leeuwenhoek in 1715.
Leeuwenhoek and the microscope
leeuwenhoek, Dutch optician and naturalist, is recognized as the inventor of the optical microscope; therefore, the one who was the first to observe, acutely and precisely, some natural phenomena such as the circulation of red blood cells in the capillaries, the existence of male germ cells, the first exact identification of the lamellar structure of the lens, the discovery of protozoa and bacteria called “small animals”. Obviously it wasn't just the availability of the best optical lenses of the time, which he also built himself.
This is how Leeuwenhoek wrote in a letter to the Royal Society of London in 1692, responding to criticism from some of his contemporaries:
I know very well, Honorable Gentlemen, that the reports I write and send you from time to time do not always agree with each other, and that contradictions can be found in them; by which I want to say once more that I am in the habit of sticking to the data I have until I am better informed or until my observations lead me elsewhere; and I will never be ashamed to change my method.
This is how modern microscopy was born, that is, the study of nature in small quantities, which still today constitutes one of the main means of investigation of modern scientific research. But to better understand the birth and evolution of this science, we must refer to the numerous intuitions and discoveries that from the first attempts in Antiquity have shaped the development of this discipline to the astonishing observations of modern science.
Light in the Hellenic and Islamic tradition
Although the microscope is a relatively recent invention, the study of light phenomena has interested many of the great minds of Antiquity and has given rise to debates between different schools of thought; We already owe it to great thinkers such as Aristotle, or Euclid, who lived between the IV and III centuries BC, whose first formalization of which we have written evidence of the concept of vision and rays of light. Already in the XNUMXrd century BC. C. the use of the famous burning mirrors of Archimedes became famous during the Second Punic War, although it has not yet been historically proven.
Roma
The most documented examples in this regard are those that come from the roman world. In fact, the use that the ancient Romans made of more or less flattened glass spheres to concentrate the sun's rays and obtain fire has been widely accredited for a long time. Lens technology seems to be even older than Roman civilization, as evidenced by the finds from Knossos, which date back to the Bronze Age, a period between 3500 and 1200 BC. c.
Pompeii
In addition to crystal prisms of extraordinary precision and regularity (used to break light into the colors of the spectrum), they also come from the excavations of ancient pompeii small round vessels, slightly convex, capable of providing a clear and magnified image. Unfortunately, there are almost no literary sources that speak of these objects as vision tools. It was handed down by Pliny the Elder when the emperor Nero, perhaps myopic, used to watch gladiator fights by looking at them through a large polished emerald.
Ottics and Catoptrics
Returning to Euclid, we note that he was the author of the famous five postulates of geometry that contain the concepts of point, line and plane; These fundamental concepts came together in the work Ottica e Catoptrica where elements of perspective are contained, the study of reflection in plane and spherical mirrors and, for the first time, the concept of visual ray without physical structure is defined. This allows Euclid to extend the typical method of geometric demonstrations to the field of light phenomena.
The nature of these axioms, however, is strongly conditioned by the idea that vision takes place by rays emitted by the eye: the extromisive theory of light. To arrive at a more advanced theory of vision, it was necessary to wait until the 965th century, with the theories of the Arab Alhazen (1039-XNUMX). According to Alhazen, the eye cannot "feel" the object except by means of rays which sends you with a finite velocity; the light must have a real existence because when it is very intense it can damage the eyes and generate secondary images.
The invention of the microscope
It will be necessary to wait until the Baroque era to see the birth of the true precursor microscope of the modern ones. The 1609th century is a fruitful period in many countries for science in general, in fact it must be said that it saw a true scientific revolution with Bacon, Boyle, Copernicus, Leibniz and many others. However, it must be said that in the history of microscopy there is no outstanding date comparable to XNUMX, the year in which Galileo Galilei (1564-1642) was made with a rudimentary telescope.
cloth makers and microscopes
Furthermore, it is no coincidence that the Netherlands was the cradle of an instrument such as the microscope, since in the XNUMXth century this country represented an important commercial crossroads for the textile sector and, at the same time, for the production of ceramics and majolica. From these last workshops, perhaps as a secondary product of the manufacturing process, in all probability came the drops of molten glass that fabric producers used as small magnifying glasses to better control the texture during the production phase. This was the first use that Antoni Van Leeuwenhoek (1632-1723), initially a fabric store manager, made of solidified glass beads; later, probably following his interest in the natural sciences to which he was naturally inclined.
Therefore, Van Leeuwenhoek's can be considered the first microscope, since it was specially conceived and optimized for use for scientific research purposes. Not surprisingly, he was cited at the time as the brilliant researcher who
[…] has designed microscopes that far exceed those seen so far…
In fact, Leeuwenhoek's microscope consists of a single lens mounted on a metal support equipped with a special sample holder with adjustable focus by means of a screw mechanism, and provides for the use of artificial lighting. These elements, in addition to constituting, from that moment, the foundations of any optical microscope, presuppose a methodology for the study of natural phenomena with an already modern flavor.
Arcana Nature
Leeuwenhoek was covered with official recognition, his laboratory was visited by academics and political figures from all over the world (the famous visit of Tsar Peter the Great of Russia). Leeuwenhoek died at the age of 91, on August 26, 1723, after having seen the Latin edition of the complete collection of his numerous letters and reports, published in 1722 under the title of "Arcana Naturae."
The efforts of scholars in the following centuries will be devoted entirely to building more powerful microscopes and to systematizing, classifying, and quantifying the newly discovered microworld. In this sense, the contribution of the Englishman Robert Hooke (1635-1703) is fundamental, more remembered for his studies on elasticity than for those of optical microscopy. Hooke, a complete scholar, made improvements to the microscope, fitting it with new optical systems and a new illumination system. This allowed him to make a series of discoveries, such as the cavities in the cork, separated by walls, which he called cells. In polemics with Isaac Newton, probably the greatest scientist of the time, he supported the idea of a wave theory of light as opposed to the corpuscular theory.
The evolution of microscopy between the XNUMXth and XNUMXth centuries: from the optical microscope to the electron microscope
The improvements gradually introduced in the compound microscopes built in the XNUMXth century were essentially concerned with the mechanical structure. Although some progress had been made in the lens manufacturing techniques, the optical performance was still poor. This was due to both the quality of the glass and two serious flaws in the lenses: spherical aberration and chromatic aberration, which resulted in blurry and iridescent images.
Furthermore, each improvement always and only took place on an empirical basis and therefore They were handmade products.. To be corrected, these aberrations require the coupling of several lenses and, therefore, it was not until the middle of the XNUMXth century that such systems could be realized.
Ernst abbe
From that moment on, theoretical studies and technological progress went hand in hand. The most representative figure of this period was the German Ernst Abbe (1840-1905), who transformed the microscope from a qualitative to a quantitative instrument; many of the principles on which the modern technology of microscope optics and lenses in general are based are due to him; Abbe collaborated with Carl Zeiss (1816-1888) in the famous Jena optical workshops.
He derived the expression, which bears his name (Abbe number), to characterize the dispersive power of glass and related the resolution of a microscope objective as a function of its numerical aperture. many of the principles on which the modern technology of microscope optics and lenses in general are based are due to him. Abbe collaborated with Carl Zeiss (1816-1888) in the famous Jena optical workshops.
August Kohler
From 1900 August Kohler (1866-1948) also worked in Jena, who dealt with microphotography and perfected a now universally adopted illumination system for microscopes; At the end of the XNUMXth century, excellent straight and inverted instruments already existed on the market.
In 1903 Richard Zsigmondy (1865-1929) developed the so-called ultramicroscope, which allows the study of colloidal particles with dimensions smaller than the wavelength of light; and in the decades that followed the pace did not slow: new techniques such as phase contrast, interference methods and reflection microscopy They opened up new fields of application while other well-known techniques were perfected, such as fluorescence, contrast interference and polarization. radiation.
electron microscopy
Already in the 30s, with the definition of elementary particles such as the electron and the introduction of the wave/particle dualism to explain their behavior, the times were ripe because the limits on the spatial resolution of optical microscopes, imposed by the wavelength of light, could be surpassed in the context of a completely new perspective: electron microscopy. The first electron microscope was built in 1933 by the German physicists Ernst Ruska (1906-1988) and Max Knoll (1897-1969). Ruska himself, many years later, would refer to those times as a fruitful period of study and research:
After his graduation (1931), the economic situation in Germany had become very difficult and it did not seem possible to find a satisfactory position at the university or in industry. Therefore, I was pleased to be able to continue my activity free of charge as a PhD student at the High Voltage Institute…” .
Late XNUMXth century and scanning probe microscopy
It is still the progressive systematization of the laws of quantum mechanics that suggests new solutions to investigate the microscopic world in ever greater detail, even going so far as to reveal its intimate nature, that is, molecules and atoms. Unlike what happened before, in the 1980s some great ideas were developed in contexts that were already intellectually open and, what is not too bad, adequately endowed with human, technological and economic resources.
George Gamow
It is from the idea of George Gamow (already discoverer of the so-called Cosmic Background Radiation) of the existence of the tunnel effect, formulated in 1928, that two German physicists, Gerd Binnig (1947) and Heinrich Rohrer (1933-2013) conceived in 1981, while working at the IBM research laboratories in Zurich, the first scanning tunneling microscope.
This microscope uses a fine needle probe to detect a weak electrical current between the probe and the surface of the sample being studied, which can be investigated to a resolution theoretically smaller than the size of atoms and molecules. This discovery earned its discoverers the 1986 Nobel Prize in Physics. It is quite remarkable that the prize was awarded, rather late, to Enrst Ruska as well. "For his fundamental work in electron optics and for the design of the first electron microscope".
scanning microscopy
In the same context, but based on the electrical force exerted by the atoms of a surface on a small probe placed nearby, the Atomic Force Microscope was invented (1982) (with the collaboration of Binning himself), whose creation relies on the joint contribution of other scholars, including Calvin Quate (1923-2019) and Christoph Gerber (1942). This microscope made it possible to extend the application of scanning probe microscopy to a wide category of samples, including biological ones.
Due to its wide range of variants and applications, this technique is today, in all probability, the most versatile for the study of surfaces in the field of nanotechnology. Today, in fact, microscopies aim to obtain more and more complete information on the nature of surfaces and modern microscopes integrate, in the same instrument, different techniques to adapt to the study of samples of different nature.
From the renaissance of optics to the nanoscope
The development of laser sources that took place in the second half of the XNUMXth century represented a new development of a more classical optical field, in fact it can be said that it constituted the most important discovery in optics after that of X-rays. The characteristics of laser light (extreme coherence, high intensity and single wavelength) allow avoid phenomena of aberrations and diffractions characteristic of the light produced by traditional incandescent lamps.
In 1955, on the occasion of his doctoral thesis in mathematics, Marvin Lee Minsky (1927-2016), one of the founders of artificial intelligence, theorized about the confocal microscope, an optical instrument with unprecedented resolution and image quality for epoch. As he himself says:
In 1956, I patented my confocal microscope, but the patent expired before anyone built a second one. We didn't even bother to patent the screen or the logo, thinking they were totally obvious inventions. It seems that the obvious is not relevant to the patent.
confocal microscope
A confocal microscope differs structurally from the traditional fluorescence microscope by the use of the laser source but above all by the presence of a diaphragm along the optical path that allows to exclude the signal coming from the portions above and below the focus of the sample, thus providing an image for the first time with three dimensional information. In reality, the confocal microscope enters laboratories only in the late 80s when laser and computer technology become relatively accessible and powerful enough. It is currently a fundamentally important tool in biomedical scientific research.
The confocal microscope represents, for the field of optics, not a technological goal but a starting point for the flourishing of new research techniques based on laser technology and the use of new fluorescent markers, such as TIRF (Total internal Reflection Fluorescence) microscopy, Live Cell Imaging, confocal spectral microscopy, the use of different imaging techniques, morphofunctional analysis including FRAP (Fluorescence Recovery After Photobleaching), FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging), FCS (Fluorescent Correlation Spectroscopy) and finally the use of multiphoton lasers to obtain a significant increase in power penetration of light into the sample.
STED microscopy
The early years of this century are also marked by the development of ingenious new ideas that have pushed optical resolution beyond the limits imposed by the nature of light. In fact, we are talking about super resolution, achieved thanks to three main different approaches: lSTED microscopy developed by Stefan Hell (1962), Nobel Prize in Chemistry in 2014, structured light microscopy that owes its birth to Mats Gustafsson (1960-2011). ), and localization microscopy, introduced in the Harvard laboratories by Xiaowei Zhuang (1972), capable of visualizing a single molecule with a resolution 10 times greater than traditional optical microscopy.
The introduction of super-resolution techniques led to modern light microscopes, which can therefore reasonably be called "nanoscopes". dialogue more and more with electronic microscopes for a better integration of morphological analyses. Today, the microscope is an irreplaceable tool in the laboratory and has become the very symbol of scientific research.
The future of microscopy
The microscope was undoubtedly one of the greatest revolutions in the history of science, marking the birth of microbiology, cytology, and cell biology. The giant leaps that medical research has taken in the last 100-150 years, with all that has followed, would have been unthinkable without the microscope.
The new frontiers of technology already see the marriage between the information produced by microscopes and the use of artificial intelligence. This new discipline, called Deep Learning, is capable of analyzing images taken with microscopes and can radically change microscopy and pave the way for new discoveries. But Mats Gustafsson, one of the fathers of super resolution, had already realized all this when he said: “Once a computer is added between the microscope and the human observer, the whole game changes. At that moment, a microscope is no longer a device that must generate a directly interpretable image. Now it is a device for recording information.”
At this point, it would be legitimate to ask how far it is possible to go in the investigation and study of microscopy: the microscopic world constitutes an almost inexhaustible reservoir of information: matter possesses structural, chemical, and physical properties that reflect the imprint given by the fundamental constants and the homogeneity of physical laws arose in the first moments of the Universe and the possible variants, most of which are still beyond our comprehension, constitute the unimaginable variety of the world that we observe.
