Characterization of optical-aberration-induced lateral and axial image inhomogeneity in multiphoton microscopy

The effects of off-axis optical aberration in multiphoton microscopy and the resulting lateral and axial image inhomogeneity are investigated. The lateral inhomogeneity of the scanning field is demonstrated by second harmonic generation (SHG) imaging of fasciae and two-photon fluorescence (TPF) microscopy of thin fluorescent samples. Furthermore, refractive index mismatch-caused intensity attenuation of the TPF signal at central and peripheral regions of the scanning frame is measured using homogeneous 10-microM sulforhodamine B samples with refractive indexes of 1.33 and around 1.465. In addition to characterizing image field convexity, we also found that image resolution degrades away from the optical axis. These effects need to be accounted for in both qualitative and quantitative multiphoton imaging applications.