Description of deep saturated excitation multiphoton microscopy for super-resolution imaging

Here we recount the standard two-level model that describes saturated excitation (SAX) in multiphoton microscopy (MPM), a new technique for super-resolution fluorescence microscopy in scattering tissue, which requires no special chemistry and only simple modifications to a commercial MPM microscope. We use the model to study conditions required for improvements in MPM SAX resolution and experimental implementation strategies. Simulation results find zeros, or nodes, in the frequency response, which generate highly irregular point-spread functions (PSFs), such as rings and ripples, that contain spatial frequency content >3× larger than allowed by diffraction. These PSFs are a direct result of zeros in the frequency response of saturated fluorophores under specific excitation conditions. The impact on image quality is discussed using simulations of targets imaged with SAX PSFs. Further, we explore engineering sets of irregular PSFs by varying the excitation power and reconstructing super-resolution images from the set of captured images.