From Single-Molecule Spectroscopy to Super-Resolution Microscopy: Super-Resolution Optical Fluctuation Imaging and Metal-Induced Energy Transfer

Abstract

The presentation gives and overviews of two super-resolution fluorescence microscopy techniques are presented which are inspired by single-molecule spectroscopy. The first is Superresolution Optical Fluctuation Imaging (SOFI), which converts temporal intensity fluctuations of emitters into an enhanced spatial resolution of an image. Besides providing higher resolution, SOFI is also very efficient in suppressing any background or auto-fluorescence, increases image contrast, and allows for recording three-dimensional images (z-sectioning) with a wide field microscope. The fundamental principles behind SOFI as well as several applications for super-resolved, three-dimensional cell imaging will be presented.The second technique is Metal-Induced Energy-Transfer Imaging. When placing a fluorescent molecule close to a metal, its fluorescence properties change dramatically. In particular, one observes a strongly modified lifetime of its excited state (Purcell effect). We call this effect metal-induced energy transfer or MIET. The MIET-coupling between an excited emitter and a metal film is strongly dependent on the emitter's distance from the metal. We have used this effect for mapping the basal membrane of live cells with an axial accuracy of ∼3 nm. The method is easy to implement and does not require any change to a conventional fluorescence lifetime microscope; it can be applied to any biological system of interest, and is compatible with most other super-resolution microscopy techniques which enhance the lateral resolution of imaging. Moreover, it is even applicable to localizing individual molecules, thus offering the prospect of using single-molecule localization microscopy for structural studies of biomolecules and biomolecular complexes.