RFP-Cyanine Dye Probe Pair forIn vivoNeurovascular Multiphoton 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 neuron network and blood vessels in deeper brain layers in a non-invasive manner remains to be a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2P and 3P fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum around 550 nm is used as a 3P excited probe to label neurons, and a cyanine derivative dye Q820 has a NIR absorption maximum of 825 nm as a 2P excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging. In particular, the two-photon cross section of Q820 was found to be about 2-fold higher than that of indocyanine green (ICG), a commonly used red two-photon fluorescence labeling agent, at the same excitation wavelength. Benefited 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 mouse brains through white matter and deep into the hippocampus in somatosensory cortex.