STED microscopy has gained recognition as a method to break the diffraction limit of conventional light microscopy.
Despite being a new technique, STED is already successfully implemented in life science research. The resolution
enhancement is achieved by depleting fluorescent markers via stimulated emission. The performance is significantly
dependent on the laser source and the fluorescence markers. Therefore the use of novel fluorescent markers in
conjunction with the right laser system was the main focus of our research. We present new developments and
applications of STED microscopy, unraveling structural details on scales below 90nm and give an overview of required
specifications for the solid state laser systems.
Modern microscopy in life sciences is ruled by development and exploration of new dyes and stains (probes for histochemical staining, quantum dots, fluorescent proteins etc.) on one side, and technological improvements and innovations for fluorescence microscopy-especially high resolution and optical sectioning microscopy-on the other side. Concerning the technical innovations, several ingenious inventions have been made available for confocal microscopy. First, the acousto optical tunable filter, which allows switching and dimming of laser lines. Second the spectral detector, employing mirror sliders in front of the detectors which allow continuous tuning of the spectral emission band detected by the sensor. Third, the most challenging task: a substitute to the classical beam splitter-the device which is restricting fluorescence microscopy most. This was solved by introduction of the acousto optical beam splitter. The very last device which is still lacking flexibility is the laser source, operating only at non-equidistant frequencies and requiring a set of quite different laser sources as gas lasers, solid state lasers or diode lasers. A new approach by supercontinuum light sources is presented and discussed, which significantly enhances flexibility and coverage of the excitation spectra of typical, rare and natural fluorochromes.
Due to the tendencies in biological research towards multiple stainings and increased use of newly developed dyes like fluorescent proteins, the application areas of multispectral confocal imaging increase rapidly. We present here the most recent advances in this technology particularily concerning but not limited to the excitation module, the detection module, and the beamsplitter optics of multispectral confocal microscopes.