for Ultrafast Multiphoton Microscopy

ABSTRACT As ultrafast multiphoton microscopes become more useful for biological imaging, a major challenge for researchersis to determine the exposure conditions that provide the best combination of image resolution, contrast and specimenviability. To do this requires an accurate understanding of the spatial and temporal evolution of ultrashort pulses atthe focus produced by a microscope objective. The objective itself, however, can significantly alter the pulses.Some effects, such as the broadening of pulses due to group delay dispersion in materials along the path, are understood and partial compensation for them can be made. Other effects, such as radial variations in the propagation time and variations in the pulse width, are less well understood. In this work, we investigate theradially dependent propagation and focusing of ultrashort pulses through a Zeiss CP-Achromat lOOx, I .25 NA,infinity-corrected, oil immersion microscope objective. We also extend to this high numerical aperture case thetechnique of collinear type II second harmonic generation frequency-resolved optical gating which has previouslybeen used to measure the temporal intensity and phase of ultrashort pulses at the focus of air objectives with lowernumerical aperture.Keywords: ultrashort pulse, multiphoton microscopy, frequency-resolved optical gating