Effect of a finite-size pinhole on noise performance in single-, two-, and three-photon confocal fluorescence microscopy

It is known that signal level in single-, two- and three-photon confocal fluorescence microscopy increases with the size of the detector. Here we evaluate the signal-to-noise and the signal-to-background criteria for these microscopes. We investigate the effect of pinhole size on their ability to detect a weakly fluorescent point object in the presence of a uniformly fluorescence background. Numerical results based on a paraxial approximation theory show that optimization of these criteria gives an optimal value for pinhole size, which results in an improved imaging performance. The resulting improvement in noise performance, compared with the use of a large detector, is greater for three-photon than for two-photon confocal fluorescence microscopes.