Morgridge Institute for Research, Madison, Wisconsin, United States.
University of Wisconsin-Madison, Department of Biomedical Engineering, Madison, Wisconsin, United States.
J Biomed Opt. 2021 May;26(5). doi: 10.1117/1.JBO.26.5.056502.
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.
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].
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.
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.
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.
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无需标记即可定量细胞代谢的方法。然而，细胞外 pH 对黄素寿命的影响目前尚不清楚。
定量细胞外 pH 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）[NAD（P）H]荧光寿命之间的关系。
将人乳腺癌（BT474）和宫颈癌细胞（HeLa）置于 pH 调节的培养基中。使用双光子荧光寿命成像技术获取细胞内 pH 指示剂或内源性荧光的图像。在相同的 pH 范围内定量测定溶液中 FAD 和 FMN 的荧光寿命。
在两种细胞系中，细胞内和细胞外 pH 之间的关系呈线性。在细胞外 pH 4 到 9 之间，FAD 的平均寿命随 pH 的增加而增加。NAD（P）H 的平均寿命随细胞外 pH 在 5 到 9 之间的增加而降低。NAD（P）H 寿命与细胞外 pH 之间的关系在两种细胞系之间存在差异。FAD、FAD-胆固醇氧化酶和 FMN 溶液的荧光寿命分别随 pH 的增加而降低、无趋势和无趋势。
内源性荧光寿命随细胞外 pH 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。
