Characterization of multiphoton microscopes by the nonlinear knife-edge technique

Imaging submicron fluorescent microspheres are the standard method for measuring resolution in multiphoton microscopy. However, when using high-energy pulsed lasers, photobleaching and heating of the solution medium may deteriorate the images, resulting in an inaccurate resolution measurement. Moreover, due to the weak higher-order response of fluorescent microspheres, measuring three-photon resolution using three-photon fluorescence (3PEF) and third-harmonic generation (THG) signals is more difficult. In this report, we demonstrate a methodology for complete characterization of multiphoton microscopes based on second- and third-harmonic generation signals from the sharp edge of GaAs wafers. This simple methodology, which we call the nonlinear knife-edge technique, provides fast and consistent lateral and axial resolution measurement with negligible photobleaching effect on semiconductor wafers. In addition, this technique provides information on the field curvature of the imaging system, and perhaps other distortions of the imaging system, adding greater capability compared to existing techniques.