Real-time photoactivated SRS imaging enables spatiotemporally controlled nitroxyl release and therapeutic guidance in vivo

Quantitatively tracking the dynamics of reactive species in vivo with reliable spatiotemporal resolution is essential yet challenging, as it provides critical insights into physiological mechanisms, optimizes drug delivery, and advances precision medicine. Herein, leveraging stimulated Raman scattering (SRS) for in vivo quantification, this work develops a series of versatile photoactivable scaffolds that efficiently generate equivalent amounts of reactive nitroxyl (HNO) and cyano-azobenzene with high photoconversion efficiency (95.1%) and fast reaction dynamics (t1/2 = 3.97–33.56 s). Upon photoinduction, the cyano-azobenzene serves as a sensitive SRS imaging reporter, enabling precise monitoring of HNO release with high spatial resolution. Moreover, the modularly designed scaffolds ensure tuneable reaction kinetics and distinct vibrational fingerprints (2224–2245 cm−1) for multiplexed SRS imaging, along with specified organelle-targeting capabilities. By tracking the photo-release of HNO in living cells and zebrafish using SRS imaging, we uncover kinetic lags in photo-triggered HNO, confirming its distinct spatiotemporal dynamics in complex living systems. Furthermore, this study monitors the therapeutic process of photoactivated HNO in a zebrafish model of heart failure using SRS, providing a robust strategy for quantifying reactive species in native biological environments. Stimulated Raman scattering (SRS) microscopy is a powerful label-free bioimaging technique for real-time monitoring of photo-controlled drug delivery and metabolic processes. However, current SRS probes lack quantitative capability for tracking released species with high spatial resolution. Here, the authors present a series of photoactivatable SRS probes that enable quantitative, real-time visualization of bioactive HNO release with robust spatiotemporal resolution in a zebrafish model.