Developments in fluorescence nanoscopy

Abstract

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 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) microscopy3,4. 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 activated, 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.