A novel nanoscopic tool by combining AFM with STED microscopy

The classical diffraction limit for the resolution of an optical microscope can be overcome (Hell [2009]) and that was demonstrated in the last years. Super-resolution techniques based on stimulated emission depletion (STED) and others (Schermelleh et al. [2010]) showed their potential in material science applications and biological sciences (Willig et al. [2006]). Nowadays, most of these techniques are commercially available and are routinely used on live biological samples (Pellett et al. [2011]). The most inspiring aspect for all these ultrahigh resolution approaches is the promise of theoretically unlimited resolution (Hell [2003]).The basic idea of STED is to selectively switch off the periphery of the fluorescent focus by superimposing the diffraction limited excitation focus with a second beam, red shifted in wavelength with respect to the excitation wavelength. This second beam stimulates the excited dye molecule to release its energy across an energy transition which corresponds to the same energy, i.e. same wavelength, like the stimulation beam itself. Hence the fluorescence emission is inhibited in those areas where the second beam falls and the dye molecule appears dark to the detector. By adding an appropriate phase mask, the focal distribution of the stimulating beam can be tuned into a doughnut-like shape featuring zero intensity at its center. Now, exciting the molecules with a regular excitation laser beam and de-exciting them to its ground state with a donut shaped STED laser beam leaves fluorescent molecules in an area of sub-diffraction dimensions. Saturating the stimulated emission depletion process by increasing the power of the STED beam further reduces the size of the fluorescing area and thus attains ‘super resolution’. In contrast to any currently used super resolution techniques, STED microscopy possesses a unique set of advantages, such as, rapid image acquisition that enables to study fast dynamics; for example, vesicle movement in vivo,