Photonics (May 2023)

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,
  • Alberto Diaspro

Journal volume & issue
Vol. 10, no. 5
p. 601


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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.