Dispersion compensation in three-photon fluorescence microscopy at 1,700 nm.

Signal generation in three-photon microscopy is proportional to the inverse-squared of the pulse width. Group velocity dispersion is anomalous for water as well as many glasses near the 1,700 nm excitation window, which makes dispersion compensation using glass prism pairs impractical. We show that the high normal dispersion of a silicon wafer can be conveniently used to compensate the dispersion of a 1,700 nm excitation three-photon microscope. We achieved over a factor of two reduction in pulse width at the sample, which corresponded to over a 4x increase in the generated three-photon signal. This signal increase was demonstrated during in vivo experiments near the surface of the mouse brain as well as 900 μm below the surface.