The cross-talk modulation of excited state electron transfer to reduce the false negative background for high fidelity imaging <i>in vivo</i>

Abstract

In practice, high fidelity fluorescence imaging <i>in vivo</i> faces many issues, for example: (1) the fluorescence background of the probe is bleached by the wide intensity scale of fluorescence microscopy, displaying an inherent false negative background (<b>FNB</b>); and (2) the dosage of the probe has to be increased to achieve sufficient intensity for <i>in vivo</i> imaging, causing a vicious cycle that exacerbates the <b>FNB</b>. Herein, we constructed a fluorophore (<b>F</b>)-electron donor (<b>D</b>)-electron regulator (<b>R</b>) system, and thereby developed a dual modulation strategy for the <i>de novo</i> design of high fidelity probes. Using cross-talk modulation, the probe allows: (1) enhanced <b>ESET</b> (excited state electron transfer) from <b>F</b> to <b>D</b>, which minimizes the inherent <b>FNB</b> based on synergistic <b>PET</b> (photo induced electron transfer); and (2) the inhibition of <b>PET</b> and weakening of <b>ESET</b> from <b>F</b> to <b>D</b> to maximize the reporting intensity to further reduce the <b>FNB</b>, which is additionally enhanced by an overdose of the probe. To test the implementation, we constructed a 7-hydroxy-2-oxo-2<i>H</i>-chromene-3-carbaldehyde (<b>HPC</b>) series of probes, with HPC (<b>F</b>) as the fluorophore, 2-hydrazinylpyridine, which was screened as an electronically adjustable donor (<b>D</b>), and electronic regulators (<b>R</b>). In particular, <b>HPC-7</b> and <b>HPC-8</b> provided cell/zebrafish imaging with negligible background even using the rather low fluorescence scale of microscopy (a region for revealing hidden background). Interestingly, with the specificity of <b>HPC</b> for reporting zinc, we achieved probe <b>HPC-5</b>, which possesses both an ultralow inherent <b>FNB</b> and optimal reporting intensity for tissue and <i>in vivo</i> imaging, enabling the <i>in vivo</i> imaging of zinc in mice for the first time. Under this high-fidelity mode, the fluorescence monitoring of zinc ions during the development of liver cancer in mice was successfully performed. We envision that the dual modulation strategy with the <b>F-D-R</b> system could provide a useful concept for the <i>de novo</i> design of practical probes.