Developments in fluorescence nanoscopy

Far field optical microscopy and especially confocal fluorescence microscopy are well established methods for the non-invasive 3D-investigation of cellular structures. However, the resolution of conventional light microscopy is limited by diffraction to ~200nm in the focal plane and ~600nm along the optic axis¹. In order to discern identical labels which are much closer than this, one has to overcome the diffraction barrier. The utilization of optical switching events allows one to circumvent Abbe\´s diffraction limit²: The switching of only markers within an area which is much smaller than the size of a diffraction limited spot to a visible "bright" state while all other markers are switched to a non-visible "dark" state defines a sub-diffraction area. By sequentially recording all areas within the diffraction spot, it is possible to assemble a sub-diffraction image. The first radical concept for improving the resolution of a far field microscope was Stimulated Emission Depletion (STED) microscopy^3,4. In this concept the saturated depletion of the excited state of the fluorescent molecule is used to generate a fluorescent spot that is narrower than the diffraction limit. In biological samples, spot diameters of 15-20nm have been demonstrated⁵, meaning that the resolution is higher by more than an order of magnitude as compared to confocal microscopy. In principle, the resolution of a STED-microscope can be increased down to the size of a molecule. An other method utilizing molecular switching events for achieving nanoscale resolution in microscopy uses a more pointillist approach. For example photoactivated localization microscopy (PALM)^6,7, stochastic optical reconstruction microscopy (STORM)⁸ and PALM with independently running acquisition (PALMIRA)⁹ all bear different names but are based on the same principle: Single molecules which are initially in a dark state are sequentially activa- ed, located and deactivated. The localization accuracy of each molecule depends, of course, on the number of detected photons per molecule and can be has high as 2 nm. Over the whole field of view, these methods provide an average resolution in the order of several tens of nanometers.