4Pi STED microscopy

The 4Pi-STED-microscope is the result of combining the two unrelated concepts of STED- and 4Pi-microscopy. Here, the fluorescent sample is placed in the common focus of two opposing lenses, but excitation and detection are performed through a single lens (4Pi mode A). The green excitation pulse is immediately followed by a red STED-pulse, which enters the focal region through both lenses inducing stimulated emission of the excited fluorescent molecules to the ground state. To permit fluorescence emission from the center but suppress it from neighbouring regions it is useful to phaseshift the STED beam to have a minimum at the center.