RESOLFT (reversible saturable optical fluorescent transitions) technology is breaking the century-old diffraction resolution barrier in conventional microscopy and allows for resolution of the size order of a dye molecule, i.e. a sharpness of one or two nanometers.
RESOLFT (reversible saturable optical fluorescent transitions) technology, a method providing molecular scale resolution with visible light and regular lenses for use in microscopy. While a number of technologies such as electron microscopy exist to visualize virus particles, subcellular structures and macromolecules, RESOLFT fluorescence microscopy for the first time enables researchers to observe these structures inside of living cells without destroying them.
a team of researchers at the Max Planck Institute for Biophysical Chemistry in Göttingen led by Prof Dr Stefan Hell invented a method to overcome this barrier. The trick is done by using fluorescence marker molecules and two different light sources.
Fluorescence markers can be excited by light to send out fluorescent light, but light also can be used to extinguish (“quench”) the fluorescence. In RESOLFT (reversible saturable optical fluorescent transitions) microscopy this principle is applied by illuminating a spot and subsequently quenching the fluorescence sent out by this spot in a way that the fluorescing area is reduced. This can be accomplished by over-saturating the quenching intensity.
As an example, the principle is applied in STED (Stimulated Emission Depletion) microscopy: A spot exciting fluorescence markers in a probe is superimposed by a doughnut-shaped quenching beam. As a result, the fluorescence is quenched everywhere in the focal spot except in the doughnut hole. By increasing the intensity of the doughnut-shaped beam, the fluorescent spot can be progressively narrowed down, in theory, even to the size of a molecule. For imaging, a probe is scanned by the ultra-sharp spot and the fluorescence intensities are then assembled by software.