Non-degenerate two-photon imaging of deep rodent cortex using indocyanine green in the water absorption window

Fluorescent dyes exhibiting peak action cross-sections within the water absorption window are generally unsuitable for in vivo two-photon fluorescence imaging using photons of the same wavelength. We show that undertaking the two-photon excitation process with two photons of different wavelengths, namely non-degenerate two-photon excitation (ND-2PE), enables imaging in the water absorption window using two spatially and temporally overlapped excitation sources at 1300 nm and 1600 nm. We explore the relative brightness spectra of indocyanine green (ICG) and assess its suitability for imaging at wavelengths susceptible to water absorption. Further, we demonstrate damage-free in vivo imaging of the rodent cortex vascular structure up to 1.2 mm using ND-2PE.