Sub-diffraction Microscopy utilizing STED (stimulated emission depletion): Optimizing parameters for resolution and application

The resolution of a fluorescence microscope is limited by diffraction. It has been shown that this diffraction limit can be broken by STED [1]. In STED-microscopy the diffraction-limited fluorescence excitation beam is overlaid with a second red-shifted STED-beam that has an intensity profile with a central minimum, quenching the fluorescence at the focal periphery. Oversaturated quenching effectively squeezes the size of the fluorescence focal area, thus improving the resolution beyond the diffraction limit. Here, we show the importance of optimizing parameters such as the depth of the central minimum of the STED-beam for achieving the highest resolution. Furthermore, the influence of scattering media such as cell or brain tissue is investigated, enabling the use of STED microscopy deep (up to 100 µm) inside scattering samples. This offers new important fields of applications for sub-diffraction microscopy. [1] Westphal, V., and S. W. Hell (2005). "Nanoscale Resolution in the Focal Plane of an Optical Microscope" Phys. Rev. Lett. 94: 143903