Spatial and temporal characterization of the focusing of ultrashort pulses by high-NA objectives for ultrafast multiphoton microscopy

As ultrafast multiphoton microscopes become more useful for biological imaging, a major challenge for researchers is to determine the exposure conditions that provide the best combination of image resolution, contrast and specimen viability. To do this requires an accurate understanding of the spatial and temporal evolution of ultrashort pulses at the 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 the radially dependent propagation and focusing of ultrashort pulses through a Zeiss CP- Achromat 100X, 1.25 NA, infinity-corrected, oil immersion microscope objective. We also extend to this high numerical aperture case the technique of collinear type II second harmonic generation frequency-resolved optical grating which has previously been used to measure the temporal intensity and phase of ultrashort pulses at the focus of air objectives with lower numerical aperture.