Important characteristics of stimulated emission depletion microscopy

Efforts to go beyond the light diffraction limit to observe molecular events have gave rise to a suite of super-resolution microscopy techniques, some of which has been commercialized. One such super-resolution microscopy technique is stimulated emission depletion microscopy (STED). In STED, point spread function engineering is used to reduce the size of the Airy disk and help achieve higher resolution. However, not all aspects of the STED technique remain clear. Specifically, in STED microscopy, there are two excitation wavelengths, one of which is infrared energy, and the other of higher energy level. More importantly, we still do not fully understand the fundamental basis on which the central point spread function (PSF) of STED microscopy could be reduced. One possibility proposed here is that the central PSF and outer donut shaped PSF exchange energy. Specifically, the central PSF is a region of relatively high energy photons confined in a relatively small region. When the larger lower energy donut PSF is constructed by optics around the central high energy PSF, there is connection between the two PSF that allows, according to the laws of thermodynamics, exchange and equilibration of energy between the central PSF and outer donut PSF. Through this, the central PSF loses energy and become sharper, thereby allowing higher resolution. On the other hand, the outer donut PSF gain energy and photons, and become larger. Optically, this sharpening of the central PSF enhances the resolution, but reduces the photons available for imaging at the central PSF, and which calls for more sensitive cameras and detectors.