Sub-diffraction limited imaging with fluorophores exhibiting emission depletion upon saturation

We present a simplified single-beam scheme for depletion-based sub-diffraction-limited imaging which allows for less restrictive illumination conditions. This is done by introducing the concept of fluorophores exhibiting emission depletion upon saturation. We discuss the circumstances under which such a depletion based process is possible, and derive the scaling of the spatial resolution utilizing this scheme. Next, we analyze the proper illumination conditions both in space and time required for sub diffraction limited imaging, and show that it is applicable only under pulsed excitation. Finally, our scheme's advantages and shortcomings relative to alternative realizations of depletion-based sub-diffraction-limited microscopy are discussed. (C) 2008 Optical Society of America