Simultaneous 2-photon and 3-photon excitation with a red fluorescent protein-cyanine dye probe pair in the 1700-nm excitation window for deep in vivo neurovascular imaging

In vivo imaging of the neurovascular network is considered to be one of the most powerful approaches for understanding brain functionality. Nevertheless, simultaneously imaging the biological neural network and blood vessels in deep brain layers in a non-invasive manner remains to a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2-photon and 3-photon fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum of around 550 nm is used as a 3-photon excited probe to label neurons, and a cyanine derivative dye Q820@BSA has a NIR absorption maximum of 825 nm as a 2-photon excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging with high emission spectral separation (>250 nm). In particular, the 2-photon action cross-section of Q820@BSA was found to be about 2-fold larger than that of indocyanine green (ICG), a commonly used red 2-photon fluorescence labeling agent, at the same excitation wavelength. Benefiting from the long wavelength advantage in reducing scattering in both 2 and 3-photon excitation of the fluorescence pairs, we demonstrated in vivo neurovascular imaging in intact adult mouse brains through white matter and deep into the hippocampus in the somatosensory cortex.