The super-resolution technology shows objects ten times sharper than a regular light microscope.
Light waves cannot represent objects that are smaller than a half wavelength (approx. 0.25 micrometre). That is what we learned about light microscopes in secondary school. Dr Bernd Rieger and Dr Sjoerd Stallinga of the department of Imaging Physics (Applied Sciences) know better. With their super-resolution technology, they can go as far as 1/20 of the length of the light wave – this is 10 times sharper. Their technology is particularly attractive for studying biological structures.
It is based on fluorescence microscopy, which was developed in the mid-1990s. In this technique, a fluorescent molecule with a smart tail is attached to an interesting cell structure. A light pulse is then used to bring the ‘fluorophore’ into a charged state. When it reverts, it emits light of another colour. This makes it possible to distinguish between incoming and outgoing light. The light source is a molecule that is quite a bit smaller than half the length of a light wave. The light source is positioned in the calculated centre of the light patch.
Rieger compares it to taking a night-time photograph of a city in the distance, in which the lights in individual houses are turned on and off in order to achieve maximum resolution.
In this technology, it is essential to avoid illuminating all of the fluorophores at once. Initially this was achieved through the successive activation of some of the molecules with UV light, fading them out with an overdose of light after activation. This worked, but it was sometimes a lengthy process, with lighting times up to 24 hours for 100,000 frames with 1,000–10,000 light points. The lighting time has since been reduced to around five minutes.
According to Rieger and Stallinga, future developments will include: shorter registration times, clearer fluorophores and 3D registrations for dynamic systems. This would make this advanced form of light microscopy more broadly applicable in molecular biology.