Significance: Autofluorescence measurements of the metabolic cofactors NADH and flavin adenine dinucleotide (FAD) provide a label-free method to quantify cellular metabolism. However, the effect of extracellular pH on flavin lifetimes is currently unknown. Aim: To quantify the relationship between extracellular pH and the fluorescence lifetimes of FAD, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Approach: Human breast cancer (BT474) and HeLa cells were placed in pH-adjusted media. Images of an intracellular pH indicator or endogenous fluorescence were acquired using two-photon fluorescence lifetime imaging. Fluorescence lifetimes of FAD and FMN in solutions were quantified over the same pH range. Results: The relationship between intracellular and extracellular pH was linear in both cell lines. Between extracellular pH 4 to 9, FAD mean lifetimes increased with increasing pH. NAD(P)H mean lifetimes decreased with increasing pH between extracellular pH 5 to 9. The relationship between NAD(P)H lifetime and extracellular pH differed between the two cell lines. Fluorescence lifetimes of FAD, FAD-cholesterol oxidase, and FMN solutions decreased, showed no trend, and showed no trend, respectively, with increasing pH. Conclusions: Changes in endogenous fluorescence lifetimes with extracellular pH are mostly due to indirect changes within the cell rather than direct pH quenching of the endogenous molecules.

中文翻译：

---

细胞外 pH 值影响代谢辅因子的荧光寿命

意义：代谢辅因子 NADH 和黄素腺嘌呤二核苷酸 (FAD) 的自发荧光测量提供了一种量化细胞代谢的无标记方法。然而，细胞外 pH 值对黄素寿命的影响目前尚不清楚。目的：量化细胞外 pH 值与 FAD、黄素单核苷酸 (FMN) 和还原型烟酰胺腺嘌呤二核苷酸（磷酸盐）[NAD(P)H] 的荧光寿命之间的关系。方法：将人乳腺癌 (BT474) 和 HeLa 细胞置于 pH 调节培养基中。使用双光子荧光寿命成像获得细胞内 pH 指示剂或内源性荧光的图像。FAD 和 FMN 在溶液中的荧光寿命在相同的 pH 范围内量化。结果：在两种细胞系中，细胞内和细胞外 pH 值之间的关系是线性的。在细胞外 pH 值 4 到 9 之间，FAD 平均寿命随着 pH 值的增加而增加。NAD(P)H 平均寿命在细胞外 pH 值 5 到 9 之间随着 pH 值的增加而降低。NAD(P)H 寿命与细胞外 pH 值之间的关系在两种细胞系之间有所不同。FAD、FAD-胆固醇氧化酶和 FMN 溶液的荧光寿命分别随着 pH 值的增加而降低、没有趋势和没有趋势。结论：内源性荧光寿命随细胞外 pH 值的变化主要是由于细胞内的间接变化而不是内源性分子的直接 pH 猝灭。NAD(P)H 寿命和细胞外 pH 值之间的关系在两种细胞系之间不同。FAD、FAD-胆固醇氧化酶和 FMN 溶液的荧光寿命分别随着 pH 值的增加而降低、没有趋势和没有趋势。结论：内源性荧光寿命随细胞外 pH 值的变化主要是由于细胞内的间接变化而不是内源性分子的直接 pH 猝灭。NAD(P)H 寿命和细胞外 pH 值之间的关系在两种细胞系之间不同。FAD、FAD-胆固醇氧化酶和 FMN 溶液的荧光寿命分别随着 pH 值的增加而降低、没有趋势和没有趋势。结论：内源性荧光寿命随细胞外 pH 值的变化主要是由于细胞内的间接变化而不是内源性分子的直接 pH 猝灭。