Dynamic saturation optical microscopy: employing dark-state formation kinetics for resolution enhancement

Abstract

Fluorescence microscopy has become one of the most rapidly developing observation techniques in the field of molecular biology, since its high sensitivity, contrast and labeling specificity together with being non-invasive fulfill the most important requirements of live cell imaging. The biggest limitation of the technique seems to be the spatial resolution which is, based on Abbe's diffraction law, restricted to some hundreds of nanometres. Recently, various approaches have been developed that overcome the limit imposed by the diffraction barrier and these methods currently lead the development in the field of fluorescence microscopy. In this contribution, we present dynamic saturation optical microscopy (DSOM)--a new technique that monitors the temporal decay of the excited singlet state due to a dark state formation. By mapping the intensity dependent decay kinetics, enhanced resolution images can be obtained. Generally, any dark state of fluorescent molecules can be employed in DSOM. Here, we focus our attention on triplet state formation.