Abstract
Laser scanning microscopy by a multi-photon process is a powerful tool for the visualization of microstructures
in living specimens because of its merits, locality of the excitation, less invasion and deep
penetration. However, the spatial resolution of this methodology is inferior to single-photon excitationbased
confocal laser scanning microscopy and recent super-resolution microscopies. On the other hand,
stimulated emission depletion (STED) microscopy, one of the most common super-resolution
microscopies, is based on a laser scanning microscopy system. Therefore, by combining these
methodologies, recently attempts of the visualization of nano-structures in biological specimens have
been successful. These methodologies potentially have both merits of deep penetration and high spatial
resolution. In this article, we describe them in detail including our attempts to develop a new twophoton
excitation STED microscopy and discuss future research and new technologies in this field.
