Jiang Yishu, El Khoury Elsy, Pezacki Aidan T, Qian Naixin, Oi Miku, Torrente Laura, Miller Sophia G, Ralle Martina, DeNicola Gina M, Min Wei, Chang Christopher J
Department of Chemistry, University of California, Berkeley, California 94720, United States.
Department of Chemistry, University of Rochester, Rochester, New York 14627, United States.
J Am Chem Soc. 2024 Dec 11;146(49):33324-33337. doi: 10.1021/jacs.4c06296. Epub 2024 Nov 25.
Molecular imaging with analyte-responsive probes offers a powerful chemical approach to studying biological processes. Many reagents for bioimaging employ a fluorescence readout, but the relatively broad emission bands of this modality and the need to alter the chemical structure of the fluorophore for 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 activity-based sensing (ABS) mode. We illustrate this concept with two copper Raman probes (CRPs), and , 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 C≡N or C≡N isotopic SRS tag with nearly identical physiochemical properties in terms of shape and size. SRS imaging with the reagents enables duplex monitoring of changes in intracellular labile Cu(I) and Cu(II) pools upon exogenous copper supplementation or copper depletion or genetic perturbations to copper transport proteins. Moreover, CRP imaging reveals reciprocal increases in labile Cu(II) pools upon decreases in activity of the antioxidant response 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 establishes a foundation for broader SRS applications in analyte-responsive imaging in biological systems.
使用对分析物有响应的探针进行分子成像为研究生物过程提供了一种强大的化学方法。许多用于生物成像的试剂采用荧光读数，但这种方式相对较宽的发射带以及为获得不同信号颜色而改变荧光团化学结构的需求可能会限制多重成像。在此，我们报告了一种通过在基于活性的传感（ABS）模式下利用受激拉曼散射（SRS）相对较窄的光谱特征来进行多重分析物成像的通用方法。我们用两种铜拉曼探针（CRPs） 和 来说明这一概念，它们分别通过铜导向的酰基咪唑（CDAI）化学与松散结合的Cu（I/II）和Cu（II）离子（即不稳定铜池）选择性反应。这些试剂使用带有C≡N或C≡N同位素SRS标签的染料支架，以铜依赖的方式标记近端蛋白质，在形状和大小方面具有几乎相同的物理化学性质。使用 试剂进行SRS成像能够对外源补充铜或铜耗竭或对铜转运蛋白进行基因扰动后细胞内不稳定Cu（I）和Cu（II）池的变化进行双重监测。此外，CRP成像显示在肺腺癌细胞模型中，抗氧化反应核因子红细胞2相关因子2（NRF2）活性降低时，不稳定Cu（II）池会相应增加。通过展示使用窄带宽ABS探针对不同氧化态的铜池进行多重成像，并确定癌症中不稳定金属营养池的变化，这项工作为SRS在生物系统中对分析物有响应的成像中的更广泛应用奠定了基础。
