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.
Fluorescence lifetime imaging (FLIM) data has consistently revealed a significant difference in mean FAD lifetime between in vivo and in vitro models. We hypothesized that the observed difference in mean FAD lifetime could be a result of environmental differences, such as differing glucose levels, oxygen levels, or pH, between the two models. We investigated the effects of environmental pH on the autofluorescence lifetime of FAD. We adjusted the pH of HEPEScontaining media using sodium hydroxide and hydrochloric acid. We then replaced the normal media of plated BT474 cells with the pH-adjusted media, allowed 20 minutes for cellular changes to occur, and then imaged the cells using time correlated single photon counting FLIM. We found that the mean lifetime of FAD increased with increased pH, resulting in a significant increase between pH 3.9, 6.2, 7.4, 9.1, and 9.5. The mean lifetime of NAD(P)H decreased at pH 3.9, 9.1, and 9.5 relative to a control pH of 7.3, and the optical redox ratio showed no significant changes except at pH 3.9 relative to a control pH of 7.3. These results suggest that the difference in mean FAD lifetime between in vivo and cell culture models could result from pH changes in the cellular environment.
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