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

It is known that signal level in single-, two- and three-photon confocal fl uorescence microscopy increases with the size of the detector. Here we eval uate the signal-to-noise and the signal-to-background criteria for these mi croscopes. We investigate the effect of pinhole size on their ability to de tect a weakly fluorescent point object in the presence of a uniformly fluor escence background. Numerical results based on a paraxial approximation the ory show that optimization of these criteria gives an optimal value for pin hole 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 mic roscopes. (C) 1999 Optical Society of America. OCIS codes: 110.1220, 110.42 80, 180.1790, 180.2520.