STED-4Pi Microscopy

Laser confocal scanning (LCS) microscopy is the technique of choice for 3D imaging of biological cells. Since resolution is limited by diffraction and the numerical aperture of the objective lens used, a practical resolution limit of the order of half a wavelength cannot be exceeded. Two different strategies have been pursued to overcome this limitation: By using the combined numerical aperture of two opposing lenses, 4Pi LCS microscopy offers a four to sevenfold improvement in the axial resolution down to ~100nm (1). Stimulated emission depletion (STED) microscopy quenches the effective focal spot by exploiting the photo physical properties of fluorescing dyes . While not limited by diffraction, the attainable resolution (~65nm, (2)) depends strongly on the available laser power and the photostability of the dyes used. The fusion of both of these independent methods in a hybrid STED-4Pi LCS microscope opens new paths for point-spread-function engineering to further enhance the imaging capabilities of such a system. Current developments in this field will be reported. 1. A. Egner et al. Proc. Natl. Acad. Sci. USA 99, 3370 (2002). 2. K. I. Willig et al. Nature, in press