Comparison of two-photon imaging depths with 775 nm excitation and 1300 nm excitation

We quantitatively compared the maximal two-photon fluorescence microscopy imaging depth achieved with 775 nm excitation to that achieved with 1300 nm excitation through ex-vivo TPM of blood vessels in the mouse brain. We achieved high contrast imaging of labeled blood vessels at approximately twice the depth at 1300 nm excitation as at 775 nm excitation. We also measured the two-photon excitation cross-sections of several commercially available fluorophores at 1220-1320 nm. We found that some of these fluorophores reveal comparable if not better cross-section values than those of the widely used dyes excited by shorter wavelength light.