An Activity-Based Sensing Approach to Multiplex Mapping of Labile Copper Pools by Stimulated Raman Scattering

Molecular imaging with analyte-responsive probes offers a powerful chemical approach to study biological processes. The most common types of reagents for bioimaging employ a fluorescence readout, but the relatively broad emission bands of this modality and need to alter the chemical structure of the fluorophore to achieve different signal colors can potentially limit multiplex imaging. Here we report a generalizable approach to multiplex analyte imaging by leveraging the comparably narrow spectral signatures of stimulated Raman scattering (SRS) in an activity-based sensing (ABS) mode. We illustrate this concept by developing two Copper Raman Probes (CRPs), CRP2181 and CRP2153.2, that react selectively with loosely-bound Cu(I/II) and Cu(II) ions, respectively, termed the labile copper pool, through copper-directed acyl imidazole (CDAI) chemistry. These reagents label proximal proteins in a copper-dependent manner using a dye scaffold bearing a 13C≡N or 13C≡15N isotopic SRS tag but otherwise having nearly identical physiochemical properties in terms of shape and size. SRS imaging with CRP2181 and CRP2153.2 enables duplex monitoring of changes in intracellular labile Cu(I) and Cu(II) pools upon exogenous copper supplementation or copper depletion, or through genetic perturbations to essential copper transport proteins. Moreover, CRP imaging reveals reciprocal increases in labile Cu(II) pools upon decreases in activity of the antioxidant response transcription factor nuclear factor-erythroid 2-related factor 2 (NRF2) in cellular models of lung adenocarcinoma. By showcasing the use of narrow-bandwidth, ABS probes for multiplex imaging of copper pools in different oxidation states and identifying alterations in labile metal nutrient pools in cancer, this work provides a starting point for the broader development of SRS for analyte-responsive, multiplex imaging in biological systems.