Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations using specially designed fluorescent labels. We describe a label-free ratiometric imaging technique for direct detection of changes in intravesicular serotonin concentration in live cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using relatively benign three-photon excitation and split it into two imaging channels, whose ratio reports the concentration. The technique is sensitive at a physiologically relevant concentration range (10–150 mM serotonin). As a proof of principle, we measure the increase of intravesicular serotonin concentration with the addition of external serotonin. In general, since emission spectra of molecules are often sensitive to concentration, our method may be applicable to other natively fluorescent intracellular molecules which are present at high concentrations.

中文翻译：

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活细胞中5-羟色胺的无标记比率成像

比例成像可以使用专门设计的荧光标记物定量测量细胞分析物浓度的变化。我们描述了一种无标记的比例成像技术，用于直接检测活细胞中膀胱内5-羟色胺浓度的变化。在较高浓度下，5-羟色胺会形成短暂的低聚物，其紫外线发射会转移到更长的波长。我们使用相对良性的三光子激发来访问紫外线/蓝光发射，并将其分成两个成像通道，其比率报告浓度。该技术在生理相关浓度范围（10-150 mM血清素）敏感。作为原理的证明，我们通过添加外部5-羟色胺来测量膀胱内5-羟色胺浓度的增加。一般来说，