"…by the help of Microscopes, there is nothing so small as to escape our inquiry; hence there is a new visible World discovered to the understanding."-Robert Hooke, 1665
After over 2 years of constant viral pandemic news coverage, it is easy to forget that we did not even know that microorganisms, invisible to the naked eye, existed before the mid-17th century. Although the possibility of their existence was debated as far back as the 6th century B.C., direct observation of microorganisms under a microscope was pivotal for the founding of the field of microbiology, which has brought forth amazing and important discoveries.
The story begins with Robert Hooke, a British scientist who is well-known for discoveries spanning physics, astronomy and paleontology. He also coined the term "cell," and was the first to publish a depiction of a microorganism-a microfungus that he observed with his microscope and documented in his book Micrographia (1665). Hooke's work paved the way for others to keep innovating in the nascent field of microscopy.
The Father of Microbiology and His Contemporaries
A decade after the publication of Micrographia, Antonie van Leeuwenhoek, a Dutch scientist often referred to as the "Father of Microbiology," became the first to observe bacteria with a microscope. His pioneering work in microscopy built on that of Robert Hooke, and helped establish microbiology as a legitimate scientific discipline during the Dutch Golden Age of Science that spanned the 17th century.
Spurred by an interest in lens-making, van Leeuwenhoek observed what he called "diertjes," or "small animals," in pondwater. He later documented the appearance of other minute structures and organisms, including muscle fibers, bacteria and red blood cells, using microscopes that he designed and built himself. Although he manufactured at least 25 single-lens scopes, only 9 have survived. They could magnify up to 275 times, an incredible feat for their time; Hooke's microscope was capable of magnification to around 50 times.
Van Leeuwenhoek's microscopes were much smaller than those we use today, and simple in their design. Most consisted of a small lens clamped between 2 flat metal plates, with the complete apparatus measuring around an inch across and 2 inches in length. The specimen was placed on a pin whose distance from the lens could be adjusted with 2 screws. This basic mode of specimen adjustment is still in use in some microscopes today, although developments have occurred in every other regard.
Van Leeuwenhoek never reported his findings in a scientific journal because scientific publishing as we know today did not exist in the 17th century. Instead, he received publication for some of his hundreds of submitted letters from the Royal Society in London. The first of these letters concerned observations of lice, mold and bees. However, when he wrote to the Royal Society about his first observations of single-celled organisms in late 1676, his reputation was called into question, as nobody knew that such organisms even existed. Fortunately, van Leeuwenhoek persisted in writing to the Society, and his findings were eventually widely accepted and even celebrated in his lifetime.
Although van Leeuwenhoek gets the credit for first observing and documenting bacteria, others had hypothesized about their existence hundreds of years prior. Jain scriptures dated as early as the 6th century B.C. propose the existence of "nigodas," tiny creatures living everywhere, including in the tissues of plants and animals. As we now know, this is not far from the truth. Scientists the world over also hypothesized that infectious diseases might be caused by invisible agents. 14th century Turkish scientist Akshamsaddin described these as "seeds that are so small they cannot be seen, but are alive."
n fact, some argue that a Jesuit priest may have been the first to observe microorganisms, before either Hooke or van Leeuwenhoek. Thanks to his work on projection, Athanasius Kircher was well-acquainted with lenses too; in 1646, he wrote that milk and vinegar were "abound with an innumerable multitude of worms." Following microscopic examination of plague victims' blood, he also speculated that the plague was caused by a microorganism, although he most likely observed blood cells rather than Yersinia pestis, the causative bacterium.
Once microorganisms were discovered, the field of microbiology was free to flourish. And flourish it did-the 19th century, in particular, was bursting with microbiological discovery, from Louis Pasteur's disproving of the theory of spontaneous generation, to Robert Koch's germ theory and the recognition that hand-washing might prevent infections in medical practice.
Light and Fluorescence Microscopy
Microscopes themselves have come a long way since van Leeuwenhoek's handmade lenses. Nevertheless, light microscopy is still a foundational technique used in many laboratories to observe the shapes and behaviors of microorganisms. Light microscopes are also used for applications such as diagnosing bacterial and fungal infections in resource-limited settings without access to PCR-based tests. However, light microscopy ultimately only allows scientists to visualize cells as they appear in natural light.
Thanks to the invention of fluorescence microscopy in the early 20th century, we can use fluorescent marker genes or stains to highlight different kinds of cells, or zero in on cellular components. For example, green fluorescent protein (GFP), a fluorescent protein originally isolated from a glowing jellyfish, is often attached to specific proteins. This allows scientists to follow their movements inside, or between, cells. The developers of GFP into such a tool-Martin Chalfie, Osamu Shimomura and Roger Y. Tsien-received the Nobel Prize in Chemistry in 2008. GFP can be leveraged for all kinds of applications, from tracking cancer cell metastasis to measuring the toxicity of pollutants and even investigating how axolotls regrow their limbs.
Electron microscopy
The magnifying power of microscopes has substantially increased since the earliest invention of the tool-today, the maximum magnification of light microscopes, as imposed by physical limits of visual light, is about 1,000 times. This magnification is sufficient for observing eukaryotic and bacterial cells clearly, as they measure between 1 and 100 micrometers (µm) across. While different kinds of electron microscopes achieve resolutions as small as 1 nanometer (nm), a thousandth of 1 µm), providing an extremely close peek at the tiniest of structures, including viruses and even individual atoms.
"We perceive only that part of nature that our technologies permit and, so too, our theories about nature are highly constrained by what our technologies enable us to observe."-Gilbert et al., 2012
Electron microscopy was invented in 1931 by a team of 2 German scientists, Ernst Ruska and Max Knoll. They realized that the physical limitations of light microscopy could be overcome if a beam of electrons, instead of a beam of photons, was fired at the subject. Transmission electron microscopy (TEM), which was invented by Ruska and Knoll, and scanning electron microscopy (SEM), an elaboration on TEM, are used in scientific fields ranging from geology to the life sciences and electrical engineering. SEM allows scientists to see the surface of their sample in 3D; classic examples of SEM images include close-ups of pollen grains and bedbugs that are often featured in science magazines. Meanwhile, TEM is used for structural analyses of very thin slices of sample in 2D and can be used to visualize cross-sections of cells and whole organisms.
Elaborations on this technique, such as cryogenic electron microscopy (cryo-EM), can provide even more insight, allowing scientists to visualize cellular structures in their 3D contexts. Cryo-EM makes use of cryogenic freezing, whereby samples are frozen in liquid nitrogen, to preserve the structure of proteins. This circumvents the need to crystallize proteins, which was a major limitation for studying certain types of proteins, such as those embedded in cellular membranes. For example, cryo-EM allowed scientists to reconstruct the SARS-CoV-2 spike protein structure in atomic resolution and determine how changes in its structure allow the virus to evade vaccine-based immunity. The developers of this technique, Jacques Dubochet, Joachim Frank and Richard Henderson, were awarded the Nobel Prize in Chemistry in 2017, showcasing its value to modern science.
In addition to allowing us to learn more about living organisms, microscopes have also enabled advances in nanotechnology. For instance, the discovery of carbon nanotubes, reported in 1991, was made possible by electron microscopy. These tubular structures of pure carbon, mere nanometers wide, can have excellent conductivity and tensile strength, making them attractive for many engineering uses in materials science and microelectronics.
New Techniques; New Horizons
Although clever thinkers posited that microbes existed centuries prior, the invention of microscopes and the insights that these tools provided about a world invisible to the naked eye were pivotal in kickstarting the wider study of microorganisms. In the recent words of Dr Dave Ng, Professor of Teaching at the University of British Columbia, "new ways of knowing lead to new ways of thinking"; with every advancement in microscopy techniques and technologies, we learn something new about the world around us-no matter how tiny.
