Abstract
We have studied physiological parameters in a living cell using fluorescence lifetime imaging of endogenous chromophores. In this study, pH dependence of the fluorescence lifetime of flavin adenine dinucleotide (FAD), that is a significant cofactor exhibiting autofluorescence, has been investigated in buffer solution and in cells. The fluorescence lifetime of FAD remained unchanged with pH 5 to 9 in solution. However, the fluorescence lifetime in HeLa cells was found to decrease with increasing intracellular pH, suggesting that pH in a single cell can be estimated from the fluorescence lifetime imaging of FAD without adding exogenous fluorescent probes.
Highlights
The imaging of fluorescence has been an indispensable tool in cell biology, and numerous fluorescent dyes have been developed for the imaging of living systems at a subcellular level
We showed that autofluorescence lifetime of NADH in the reduced form, that is one of the representative autofluorescent chromophores, in human cervical carcinoma (HeLa) cells became shorter with increasing intracellular pH, indicating that the pH in a cell can be quantitatively evaluated by fluorescence lifetime imaging (FLIM) of NADH without exogenous probes [11]
Autofluorescence spectrum and autofluorescence lifetime of endogenous flavin adenine dinucleotide (FAD) in HeLa cells were measured with different intracellular pHs
Summary
The imaging of fluorescence has been an indispensable tool in cell biology, and numerous fluorescent dyes have been developed for the imaging of living systems at a subcellular level. Cells and tissues contain endogenous chromophores exhibiting fluorescence called autofluorescence, and nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and amino acids having an aromatic moiety, such as tryptophan, are known as representative autofluorescent chromophores. These chromophores exist in an extensive variety of living systems and are related to cell functions and metabolic activities [2,3,4,5,6]. The superior feature of FLIM is that fluorescence lifetime of a chromophore remains unchanged with photobleaching and alternations of optical conditions [15,16,17,18] This is advantageous for quantitative space-resolved estimation of ion concentrations and cellular environment. We have recently investigated the photoexcitation dynamics of FAD using time-resolved fluorescence techniques and found that the fluorescence lifetimes of both the stacked and open conformations depend on the polar environment surrounding FAD [36]
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