The imaging of nanoscale structures is limited by optical resolution to a few hundred nanometers. By the physical imaging process even a single point emitter appears as a diffraction limited pattern which is known as the optical point spread function (PSF). Recently, there have been several approaches to circumvent this limitation and obtain information with a resolution of a few nanometers (STORM, PALM, STED etc).

The localization-based techniques obtain spatial information by observing the PSFs of single fluorescent molecules separated in time. Therefore, the fluorophores need to be cycled between a bright “on” and a dark “off” state, which is usually done by light-induced switching.

We in the Herten group developed a different technique by switching the fluorescent probes with chemical reactions. This method was named CHIRON (CHemically Improved Resolution for Optical Nanoscopy). In a first approach, we employed the complexation of Cu(II) to a bipyridine ligand, which reversibly switches “off” the emission of a nearby fluorophore.[1] We are currently working on probe design to make this method applicable for investigating biological problems at the nanoscale.

A different approach we develop to obtain information beyond the resolution limit evaluates the photon acquisition statistics. The approach exploits the single-photon emission of an individual quantum system, also known as photon antibunching. By measuring the number of simultaneous photon detection events, Counting by Photon Statistics (CoPS) can access both the emitter number and the molecular brightness of (single) molecules in a confocal volume.[2-4]

We could evaluate the method and prove that it is possible to count up to 20 fluorescent molecules in about 200 ms. As this method does not use any photo-physical processes like bleaching or blinking, it is feasible to measure the dynamics of e.g. protein clusters. We are currently further exploring the limits of CoPS and are starting to work on biological applications.