Effects of a Finite-sized Pinhole on 3D Image Formation in Confocal Two-photon Fluorescence Microscopy

Abstract

Abstract We investigate three-dimensional (3D) imaging properties in confocal two-photon (2-p) fluorescence microscopy with a finite-sized detector. The 3D intensity point spread function, the 3D optical transfer function (OTF), the strength of optical sectioning and the signal levels for point, planar and volume objects are calculated in order to reveal their relationships to the detector size. It is shown that confocal 2-p fluorescence imaging with a point detector gives super-resolution, compared with conventional 2-p imaging without a confocal pinhole. Although optical sectioning can be attained in the latter case, its strength is inferior to that in the confocal case. In particular, a finite-sized detector can result in negative values in the 3D OTF and reduce the effective cut-off spatial frequencies, thus degrading the capability of confocal 2-p fluorescence microscopy for 3D imaging. Unlike the single-photon fluorescence microscope, there is no missing cone of spatial frequencies in the 3D OTF for...