Fluorescein: biology’s guide dog

One of the first fluorescent molecules to be synthesised by humans was Fluorescein. The imposing German chemist Adolf von Baeyer created it in 1871, and was awarded the 1905 Nobel Prize in Chemistry for his work on dyes and aromatic compounds.

Many molecules with aromatic rings (like Benzene) absorb light at one wavelength and re-emit it at another, longer wavelength. Generally, the more aromatic rings together, the merrier. This is fluorescence: a side effect of many biological molecules that have aromatic chemistry.

Absorbing photonic energy entirely (as in photosynthesis) or producing photons from chemical energy (as in the bioluminescence of fireflies) is more practical for most organisms. But chemists and biologists the world over are very grateful for fluorescence, because it is remarkably useful for studying living things. Fluorescein was one  of the first man-made, mass-produced fluorescent molecules and is still in widespread use today.

Fluorescein is used in just about everything, for example:

  • marking where planes and rocket capsules have ditched in the sea
  • searching for leaks in water distribution systems
  • tracking cells (by attaching to antibodies, like tiny molecular hooks)

In each of these cases, one could make use of the ultra-violet range of light, beyond human perception.  Fluorescein dye absorbs this light and then emits light in the visible spectrum, marking its presence clearly. This makes it easier to sense, capture or count whatever you have attached the molecule to.

Precision dyes

Fluorescein is still used routinely, but inventive chemists have made more and more sophisticated dyes, with a broad range of increasingly complex chemical structures.

  • Some dyes have a very precise colour – a very narrow range of emission – so it is easy to distinguish multiple dyes from one another at the same time – very useful when imaging cells.
  • Others absorb light very efficiently (meaning less light is needed to make them fluoresce, important as it could be damaging to the cell or tissue).

Many of these dyes are known by their commercial names. One enterprising husband-and-wife team, Dick Haugland and Rosaria Haugland, created numerous fluorescent dyes and named them in a series called “Alexa”, after their son Alex. Their dyes were quite hydrophilic, making them more compatible with many of the demanding biological tasks scientists want to use them for.

If you look carefully around a cell biology lab, you may notice it’s crammed with a whole host of different fluorescent molecules. Each of them can be attached in different ways to specific biological molecules, making it easier for human eyes (or rather complicated digital cameras) to see how things work.

Nobel number 2

The properties of fluorescence gave rise to another Nobel prize – this time in super-high-resolution microscopy. A team of inventive biophysicists, including EMBL alumnus Stefan Hell, harnessed the ‘blinking’ properties of certain fluorophores (as useful fluorescent molecules are called) to create a kind of beacon. They noticed that some fluorophores recover from photo-bleaching in a random way, such that only a small proportion would be active in an image at any given time.

Find My Fluorophore

One can assume that for a collection of active fluorophores, a cloud of photons is emitted from a single spot, where the fundamental physics of photons means you can only a see fuzzy disc (2D) or sphere (3D) centred on the true position of the fluorophore. You can’t define a single photon’s position more precisely than its wavelength, but if you take the average of the positions of a collection of photons, you can actually get a more precise measure of the centre of this photon cloud – you can “beat the physics of light” by implicitly trading resolution in time (blinking) to gain resolution in space.

Fluorescent chemicals have been the trusty guide dogs of biological science for over a century, and are likely to continue showing us the way well into the future.