Light microscopes are essential instruments in biology and medicine. Unlike electron microscopes or scanning force microscopes, they can be used to observe biological cells deep within and, unlike X-ray microscopes, light microscopes do not kill the cells. However, it seemed for a long time that no details which are smaller than 200 nanometers could be seen using a light microscope, which is not a problem for the other microscopes mentioned above.
This limitation is caused by the resolution limit of the light microscope – it can only separate two objects from each other if the distance between them is not smaller than half the wavelength of the light that is used for imaging. Ernst Abbe discovered this resolution limit in 1873. However, if the objects are marked with fluorescent molecules, it is possible to break Abbe's resolution limit by utilizing molecular on and off states during image creation. Dr. Stefan Hell and Dr. Thomas Klar of the Max Planck Institute for Biophysical Chemistry in Göttingen successfully achieved this. Their work was honoured with the Helmholtz Prize in 2001.
In STED microscopy (STED stands for "Stimulated Emission Depletion"), which was developed by Stefan Hell and Thomas Klar, the fluorescent molecules which are attached to the object to be imaged can be in a bright or in a dark state. A focused green laser beam turns them from the dark state to the bright state, so that they become fluorescent. Normally, all of the molecules in the laser light spot, which is always larger than half the laser wavelength, are excited.
However, the two researchers found a way to greatly reduce the bright area. For this, the molecules are also exposed to a focused red laser beam, whose spatial intensity profile has been changed in such a way that its light spot is ring-formed and has a tiny dark hole in the center. If the green and the red lighted spot are superimposed, the molecules in the hole of the ring can still fluoresce. However, the molecules which lie further outside are transferred from the bright state into the dark state due to stimulated emission, suppressing the fluorescence. In this way, a fluorescent area can be created which is smaller than 20 nm in diameter.
By scanning an object with the two superimposed light spots and making the molecules lying on it luminescent in sequence, the researchers produced an image with a resolution far below Abbe's limit and details of 20 nm in size became visible. Since then, Hell and his colleagues have developed other high-resolution light microscopy procedures which do not require stimulated emission. For instance, the ring-shaped laser beam switches the molecules which lie outside of the holes from the light state to a dark state and parks them. Since different organelles inside of a biological cell can be marked with appropriate fluorescent molecules, the new procedures can be used to make images and even films of living cells which have details that have not been achieved up to now and which are invaluable for cell biology. Currently, Thomas Klar and his colleagues at the Johannes Kepler University Linz are working on transferring the principle of STED microscopy to optical lithography.
