Super-resolution imaging technology has been a powerful tool for revealing fine biological structures and functions. Its high-quality imaging always needs highly accurate labeling. Here, by exploiting the high specificity and affinity of natural substrates to transporters, we developed one set of substrate-based small molecule fluorescent probes for labeling membrane transporters. A glucose-based probe (Glu-probe) and tyrosine-based probe (Tyr-probe) were synthesized as two examples. Confocal imaging showed that the Glu-probe could label glucose transporters on live cells by being stuck into the binding site. Compared with antibody-probe labeling, the labeling advantages of the Glu-probe were revealed. High specificity of the Glu-probe or Tyr-probe was examined by a colocalization experiment and glucose replacement or amino acid (AA) blocking. The synthetic probes were also tested on imaging HeLa cells to confirm their wide labeling application. Additionally, we found that membrane transporters were mostly in the clustered state on cellular membranes, changing their assembly pattern to regulate the transport effectiveness. These results suggest that the substrate-based probes can serve as valuable tools for investigating the spatial information of membrane transporters.

中文翻译：

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开发基于基质的小分子荧光探针，用于各种膜转运蛋白的超分辨率荧光成像。

超分辨率成像技术已成为揭示精细生物学结构和功能的强大工具。它的高质量成像始终需要高度精确的标签。在这里，通过利用天然底物对转运蛋白的高特异性和亲和力，我们开发了一套基于底物的小分子荧光探针，用于标记膜转运蛋白。作为两个实例，合成了基于葡萄糖的探针（Glu-probe）和基于酪氨酸的探针（Tyr-probe）。共聚焦成像显示，Glu-probe可以通过粘在结合位点上来标记活细胞上的葡萄糖转运蛋白。与抗体-探针标记相比，Glu-探针的标记优势更加明显。通过共定位实验和葡萄糖替代或氨基酸（AA）封闭，检查了Glu探针或Tyr探针的高特异性。还对合成探针在HeLa细胞成像上进行了测试，以确认其广泛的标记应用。此外，我们发现膜转运蛋白大多处于细胞膜上的聚集状态，从而改变其组装模式以调节转运效率。这些结果表明，基于底物的探针可以作为研究膜转运蛋白的空间信息的有价值的工具。