Seeing the unseeable

Starting around the turn of the 17th century, natural philosophers using the light microscope saw things where, to the naked eye, there was nothing to see. The Englishman Robert Hooke observed pockets of air within cork, which he called cells; the Dutch scientist Anton van Leeuwenhoek saw living bacteria in pond water and cells within blood and even found “wee beasties,” as he sometimes called his “cavorting” specimens, in his own semen.

Electron microscope

In the 1930s, the German physicist Ernst Ruska developed a microscope with ultra-high resolution by using electrons, which have a smaller wavelength than light and can distinguish tinier features. Using Ruska’s new electron microscope, scientists could view structures within an individual cell, with the downside that they could not look at live cells.

Dyes and stains

In 1873, the Italian physician and scientist Camillo Golgi stained neurons using a silver compound that turned the cells black. The Spanish neuroanatomist Santiago Ramón y Cajal put Golgi’s method to fruitful use, making observations that led to the neuron doctrine, the now-accepted idea that the nervous system is composed of discrete cells. In 1886, Paul Mayer invented the hematoxylin and eosin staining procedure. Hematoxylin stains cell nuclei blue; eosin is nonspecifically attracted to proteins and gives the rest of the cell a contrasting reddish hue. The most important dyes used in light microscopy today, however, are fluorescent.

Fluorescence microscope

Though the fluorescence microscope was invented around 1910, fluorescence microscopy did not really take off until the end of the century, spurred by the development of fluorescent labels for specific biological structures. The most famous of these fluorescent tags is called green fluorescent protein, or GFP, a protein derived from jellyfish that emits green light when stimulated by blue light. [For more on the use of marine life as a source of fluorescent tags, see “In coral reefs, a treasure trove of tools”.] In the 1990s, scientists isolated the gene encoding GFP, which allowed them to engineer cells genetically so that GFP could be fused to a protein of interest for visualization with the fluorescence microscope. Microscopy’s palette expanded as scientists developed variations of GFP that fluoresce in different colors; and by labeling different structures with different fluorescent molecules that can be visualized at the same time, scientists can determine whether those structures are colocalized and potentially interacting. Fluorescent labels are not limited to proteins: they can also label DNA, lipid molecules, and carbohydrates. And efforts to break the diffraction limit would increasingly rely on these fluorescent proteins.