STED and GSDIM: Diffraction Unlimited Resolution for all Types of Fluorescence Imaging

As was pointed out by Ernst Abbe, the limit of resolution in an optical microscope is ruled by the aperture of the objective and the color of the light(19) His reasoning was based on the minimum distance d between two objects at which they can still be individually distinguished. The simple relation he found stated the distance to be proportional to the wavelength and inversely proportional to the sine of the half opening angle. Later derivations, e.g. by integration of diffraction patterns and applying the Rayleigh criterion yielded similar results. It might be worth noting that all descriptions of resolution imply an arbitrary definition of what “resolved” actually means. Therefore, one should keep in mind that all numbers given for resolution do not mean that there is a sharp edge beyond which all of a sudden nothing is visible. And it is wise not to overemphasize the numbers that are discussed in this context, especially if they show more than 2 significant digits. Nevertheless, all definitions are good for comparison of the various methods, instruments and preparations. A well-established measure is the full-width half maximum of the intensity distribution of a structure that is assumed to be sufficiently smaller than the expected resolution. This is a very simple and quick measurement. With Abbe’s formula, we can estimate a rough resolution limit for standard light microscopy. Assuming light of 550nm and a numerical aperture of 1.4, the resolution d=λ/2NA should reach ca. 200nm. If one wants to increase the resolution, a shorter wavelength or a higher NA is required. Higher NA is very costly and requires immersion and preparation media at a higher refractive index than that of glass or oil. Although many attempts have been made in that direction, no practicable and sensible method was figured out. Some good results were made with UV microscopes, where the resolution might reach 100nm. This is possible with light below 300nm, which again requires special and costly equipment, as there are not many glass types that transmit below 350nm. Besides the equipment, shorter wavelength increases scattering and is readily absorbed by many organic compounds. For similar reasons, but even harder by orders of magnitude, microscopy with x-rays did not find its way into laboratories. In contrast, the very elaborate and costly electron microscopy(2) did find many fields of application due to the fact that the improvement in resolution as compared to light microscopy is not just a factor of 2 (UV microscopy) or 10 (x-ray microscopy), but reaches a factor of more than 1000 (sub-nanometer). An entirely different approach to see smaller things than by diffraction-limited methods is near-field scanning optical microscopy. Here, a tiny glass tip is scanned over (or through) the sample. The resolution is defined by the size of the tip rather than by diffraction phenomena, enabling details down to some 30nm to be imaged. The obvious disadvantage of this method is the limited depth resolution. To obtain images, the tip has to be brought into the close vicinity of the object. Therefore, it is mostly a method for surface imaging of flat (sliced) objects. Incidentally: it is of course possible to see objects that are much smaller than the resolution limit – given they are dispersed enough to locate them as individual objects. R. Zsigmondy(3) earned the 1925 Nobel Prize in chemistry for his work on the chemistry of colloids. He used a microscope where the illumination was tilted by 90°. When looking through the microscope into a cuvette containing colloidal gold particles in low concentration, he could see bright spots – like stars in the sky. The size of the spots was that of the diffraction pattern (like in astronomy), even if the objects were much smaller. The illumination from an angle is still known as dark field-microscopy today.