Background Rejection in Two-Photon Fluorescence Image Scanning Microscopy

Colin J. R. Sheppard, Marco Castello, Giorgio Tortarolo, Alessandro Zunino, Eli Slenders, Paolo Bianchini, Giuseppe Vicidomini, and Alberto Diaspro (see publication in Journal )


We discuss the properties of signal strength and integrated intensity in two-photon excitation confocal microscopy and image scanning microscopy. The resolution, optical sectioning and background rejection are all improved over nonconfocal two-photon microscopy. Replacing the pinhole of confocal two-photon microscopy with a detector array increases the peak intensity of the point spread function. The outer pixels of a detector array give signals from defocused regions, and thus the processing of these, such as through subtraction, can further improve optical sectioning and background rejection.