Led by Stefan Hell of the Max Planck Institute for NanoBiophotonics in Göttingen, the group have been studying ways of improving fluorescence microscopy for the last 20 years, with this innovation in optical nanoscopy providing a real breakthrough in their research.
Hell, who won the Otto Hahn Prize in 2009, and colleagues that include Vladimir Belov and Christian Eggeling, have been examining the many possibilities opened up by fluorescent microscopy, which uses the phenomena of fluorescence to study the properties of both organic and inorganic substances, rather than typical methods of reflection and absorption.
Published in Chemistry – A European Journal (Gyuzel Yu. Mitronova et al, doi: 10.1002/chem.200903272), the research focused on the sensitivity of the technique, which depends on the brightness of the applied fluorescence markers, as well as its photostability.
Due to the wave properties, or diffraction, of light, the resolution of an optical microscope cannot go beyond about 0.2 micrometers. Unable to capture imaging details beyond this, Hell and his team developed a method for observing objects at the nanometer scale by sequentially turning the fluorescence of nearby molecules off by stimulated emission, a technique known as STED nanoscopy.
The idea of using stimulated emission depletion (STED) microscopy was developed by Hell in 1994, before demonstrating it experimentally in 1999. It is a technique that uses non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction, and the use of these dyes in STED nanoscopy can lead to images of high quality with respect to brightness and signal-to-background ratio.
The resolution produced by STED nanoscopy is significantly better than more traditional optical microscopes, which provides more detailed structural information.
The group has successfully synthesized a series of highly photostable and highly fluorescent dyes, compounds that emit green and orange light and are based on fluorine derivatives of the well-known rhodamine dye.
The rhodamine-based fluorine derivatives that have been synthesized are important because of their versatility – the compounds are available in hydrophilic and lipophilic forms. With the inclusion of amino reactive groups, they can be easily attached to antibodies or other biomolecules in the course of standard labeling and immunostaining procedures.
The study also demonstrated that these new dyes are able to cross cellular membranes and reach the interior of living cells, which could open up the possibility for new labeling strategies for biological systems.