How Does a Microscope Work? Beating the Limits of Light

The two are routinely confused and only one matters. Magnification is how much larger the image is; resolution is the smallest separation at which two points remain visibly separate. Beyond a certain point, increasing magnification is empty, enlarging a blurred image without adding information. The resolution of a light microscope is described by a formula Ernst Abbe worked out in 1873, which says the limit is roughly the wavelength of the light divided by twice the numerical aperture of the lens, where numerical aperture measures how wide a cone of light the objective can collect. That gives around two hundred nanometres for visible light, which is why individual bacteria are visible, why the structures inside them are not, and why viruses were invisible until a different technology arrived. Immersion oil, placed between the specimen and the objective to match the refractive index of glass, raises the numerical aperture and is the last easy improvement available.

Compound microscopes with two lenses appeared in the Netherlands around 1600 and were poor instruments, with severe colour fringing and distortion. Robert Hooke published Micrographia in 1665, with engravings of a flea, a louse and cork, in which he named the units he saw in cork cells after monastic rooms and gave biology its central word. Antonie van Leeuwenhoek, a Delft draper with no scientific training, took a different route, making single tiny spherical lenses of extraordinary quality and building simple microscopes around them that outperformed every compound instrument of the century, reaching perhaps 270 times magnification. He described bacteria, protozoa, sperm cells and red blood cells, reported them to the Royal Society in a long series of letters, and never revealed his lens-making method, which was reconstructed only recently. The compound microscope did not surpass his instruments until the achromatic lens, combining glasses of different dispersion to cancel colour fringing, was applied to microscopy in the 1830s.

Most living cells are nearly invisible in an ordinary light microscope because they are transparent, and the standard solution of staining them usually kills them. Several techniques exist to get around this:

• •Phase contrast, invented by Frits Zernike in the 1930s and winning a Nobel Prize in 1953, which converts the tiny differences in the speed of light through different parts of a cell into differences in brightness, making living cells visible without any treatment
• •Differential interference contrast, which produces a similar effect with an apparent three-dimensional relief
• •Dark field illumination, which lights the specimen from the side so that only scattered light enters the objective, making small particles appear bright against black
• •Fluorescence, in which the specimen is stained with molecules that absorb light at one wavelength and emit at a longer one, so that only the labelled structures glow
• •Confocal microscopy, which uses a pinhole to reject light from outside the focal plane, producing optical sections that can be stacked into a three-dimensional reconstruction
• •Green fluorescent protein and its relatives, which let a specific protein be labelled genetically in a living organism, so that a process can be watched as it happens

Two approaches break the Abbe limit and they do it in completely different ways. Electron microscopy replaces light with a beam of electrons, whose effective wavelength is thousands of times shorter, giving resolution below a nanometre and revealing viruses, organelles and, in the best instruments, individual atoms. The cost is that electrons require a vacuum and interact strongly with matter, so specimens must be thin, dried or frozen and usually coated, and nothing living survives, which is why electron microscopy and light microscopy remain complementary rather than competing. The other route keeps light and cheats the limit statistically. Super-resolution fluorescence techniques, recognised by the Nobel Prize in Chemistry in 2014, either switch fluorescent molecules on and off so that only a few emit at a time and each can be located far more precisely than it can be resolved, or use a shaped beam to switch off everything except a spot much smaller than the diffraction limit. Both produce images at tens of nanometres in living cells, which was considered impossible for a century.

The instrument created several fields outright. Cell theory, that all organisms are made of cells and all cells come from cells, depends entirely on it. Germ theory required seeing the organisms, and the identification of specific bacteria with specific diseases by Koch and others in the 1880s was microscopy plus staining plus culture. Histology and pathology are microscopy applied to tissue, and a diagnosis of cancer is still made by a human looking down a microscope at a stained section, a procedure that has changed less in a century than almost anything else in medicine, though image analysis is now beginning to assist. Materials science uses it to examine grain structure and failure surfaces. Cryo-electron microscopy, which freezes biological molecules so fast that the water becomes glassy rather than crystalline, has in the last decade become capable of determining protein structures at near-atomic resolution without crystallising them, which removed the main bottleneck in structural biology and took its own Nobel Prize in 2017.

Magnification without resolution produces a larger blur, and the resolution of a light microscope is limited to around two hundred nanometres by the wavelength of light, a limit Abbe formulated in 1873. Leeuwenhoek's single-lens instruments outperformed compound microscopes for a century and a half. Phase contrast, fluorescence and confocal techniques make transparent living cells visible without killing them, electron beams give sub-nanometre resolution at the cost of requiring vacuum and dead specimens, and super-resolution fluorescence beats the limit by locating individual switched-on molecules.