Resolution of light microscope：Abbe diffraction limit
The smallest distance distinguishable by human naked eye is generally about 0.1mm. Microscopes is used to identify smaller substances, such as bacteria, fungi, protists, and some subcellular structures. Scientists hope to explore even smaller objects, especially viruses. However, the Abbe formula shattered all hopes.
In 1872, the revolutionary Abbe diffraction limit for microscope was calculated by German physicists Ernst Karl Abbe. It is said that resolution of all optical microscopes has a physical limit due to diffraction, which is about half wavelength of the light illuminating the target object. This means that optical microscopes can only identify objects as small as 200 nm, since violet light has the shortest wavelength with about 400 nm among visible light. By the end of the 19th century, the finest light microscopes were already close to this limit, but they were still can't do anything about organelle ultrastructure, let alone molecules and atoms.
The Abbe diffraction limit was so formidable that even Abbe, the person who proposed it, had to sigh that all efforts were in vain, and no one could break through this physical limitation. However, what Abbe did not foresee was that merely 20 years after his death, the de Broglie wave or matter wave would propel the birth of an even more powerful tool—electron microscope.
TEM vs Light Microscope: Breaking Abbe diffraction limit
De Broglie studied history and literature before, so his mindset is not bound by traditional concepts. Since Einstein believed that light is both wave and particle, electrons should also exhibit wave-particle duality. In 1923, Louis de Broglie derived the wavelength formula of electron: p = h/λ. In 1927, electrons were proven to be a wave because researchers saw an interference in double-slit experiment. In 1931, German engineer Max Knoll and Ernst Ruska constructed the world first transmission electron microscope (TEM) with a magnification about 200. Visible light was replaced by electron beams, and glass lenses were replaced by magnetic lenses, but it was merely a rudimentary machine to verify whether an electron microscope was feasible. Two years later, an improved TEM could magnify objects up to 12,000 times. Its 50 nm resolution had already surpassed all traditional optical microscopes.
The first biological specimen photograph was taken by Ladislaus Marton in 1934. It was a 15-micron-thick slice of a sundew plant leaf. The first virus captured by an electron microscope was the tobacco mosaic virus in 1939. Illusory virus was transformed into an entity that could be directly observed. New organelles were found by scientists, such as endoplasmic reticulum, ribosome and lysosome. TEM images of Golgi apparatus ended a half-century debate about its existence. The detailed ultrastructure of organelles were also revealed by TEM, such as cristae in mitochondria and thylakoids in chloroplasts.
TEM Development: Damaged Sample, Negative Staining, and 3D Reconstruction
However, when the transmission electron microscope was actually invented, many biologists and physicists were conservative or skeptical about its application in biology. Tissues are mainly composed of lighter elements such as carbon, hydrogen, oxygen, and nitrogen (C, H, O, N) that interact with electrons weakly, and electron beams penetrate them effortlessly. Therefore, all the things are similarly bright in images. Contrast can be improved with a high dose, but electron bombardment will break chemical bonds in bio macromolecules. Vacuum and dehydration prevent blurred image resulted from electron scattering due to air, but they will change the sample structure, and what researchers see may be far from living tissue.
Yet some scientists still insisted on improving TEM technology, and heavy metal negative staining is a key technique. Heavy metals are absorbed by sample and accumulate to surface, gaps and depressions. The raised areas have fewer stains. It is difficult for electron beam to penetrate heavy metals with high electron density, and these areas exhibit darker shadows to reveal sample’s contour and details. Some damage caused by electron bombardment and vacuum is also weakened by heavy metals that act like a protective shield around the sample. By the 1950s, techniques for ultrathin sample had matured, and some complex biological structures were unveiled by TEM, such as chromosomes and mitochondria. It was an exciting era, as a single TEM image was often the prototype of a significant biological paper. Freezing is another method to reduce damage, leading to the emergence of Cryo-EM.
It soon became evident that each electron microscope image is a 2D projection. If one wants to reconstruct the 3D structure from 2D projections, many images from different angles are needed. For convenience, scientists chose the tail of phage T4 with its beautiful rotational symmetry and successfully resolved it in 1968. For asymmetric samples, the common line theory is used to relate their 2D projection to the 3D sturcture.