3D view of cells with flourescent markers
In contrast to STED, only separate, isolated marker molecules are randomly switched on at the same time. Their fluorescence is registered, and then they get switched off again automatically. In this way, the simultaneously fluorescing (switched on) markers are farther apart from each other than the minimum distance that the microscope can resolve. This is only possible using switchable molecules that emit many photons, one after the other, when switched on. If these photons are captured with a camera, the centers of the individual fluorescing dots can be distinguished. After the exposure, the molecule becomes dark again (switches off), allowing further, neighboring molecules to be photographed. This process is repeated many times, until many dots become a picture. The full distribution can be reconstructed – at a resolution not limited by the wavelength of light.
The researchers have now found a class of substances that fulfill all the requirements of this technique: rhodamine amides. At the core of these molecules lies a system of five rings. In this form, the compound is colorless and does not fluoresce. Irradiation with light induces an isomerization in which one of the rings is opened. This form of the molecule is red and can be excited several times.
Most importantly: rhodamine amides can be switched on by either a UV photon or two photons in the red part of the spectrum. This two-photon excitation can be focused onto a thin plane, which allows biological samples to be photographed layer by layer. The individual images can then be reconstructed into a single multilayer image. The resolution reached in the focal plane is far beyond the diffraction barrier (10–30 nm).