NIR in, far-red out: developing a two-photon fluorescent probe for tracking nitric oxide in deep tissue

As a pivotal signalling molecule involved in various physiological and pathological processes, nitric oxide (NO) has motivated increasing interest in the last few decades. Although a considerable number of fluorescent probes have been developed for NO imaging, the in situ tracking of this gas molecule in biological events remains a big challenge, mainly because of the relatively short excitation and/or emission wavelengths, which are subject to background interference and lowered collection efficiency in deep-tissue imaging. Herein, we report a far-red emissive (650 nm) two-photon (TP) excitable NRNO probe, using Nile Red as the TP fluorophore, for NO detection and imaging both in vitro and in vivo. The NRNO probe shows a fast (within 180 s) and specific fluorescence response toward NO with a limit of detection (LOD) as low as 46 nM. The excellent properties of NRNO enable it to sensitively detect both exogenously and endogenously generated NO in living cells. The "NIR in" and "far-red out" lights lead to improved penetrating ability, thus endowing the probe with high resolution for the illumination of deep tissues. It is therefore able to visualize the NO generation in a lipopolysaccharide (LPS)-mediated inflammation process for the first time. Our results demonstrate that NRNO could be a practical tool for studying the NO-related biological events. Moreover, this study also suggests the possibility of using Nile Red and its derivatives to develop far-red emissive TP probes, which is an important, yet undeveloped area.