Effect of a confocal pinhole in two-photon microscopy

We investigated the effect of a finite-sized confocal pinhole on the perfor mance of nonlinear optical microscopes based on two-photon excited fluoresc ence and second-harmonic generation. These techniques were implemented usin g a modified inverted commercial confocal microscope coupled to a femtoseco nd Ti:sapphire laser. Both the transverse and axial resolutions are improve d when the confocal pinhole is used, albeit at the expense of the signal le vel. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large. (C) 1999 Wiley-Liss, Inc.