Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence.

Aziz Ul Rehman,Martin E Gosnell,Ewa M Goldys,Ayad G Anwer,Guozhen Liu,Saabah B Mahbub

doi:10.1364/boe.8.001488

Abstract

Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence.

Highlights

Cellular processes rely on energy produced during metabolism and a number of methodologies have been developed for its characterization
We excited the NADH in the range 280-380 nm and acquired its emission spectra between 400 and 550 nm. These ranges were selected to overlap with the free NADH absorption range from 300 to 380 nm and its fluorescence range from 420 to 480 nm described in the literature [29, 30]
The excitation emission matrices (EEMs) for 50 μM NADH show that maximum of the excitation spectrum is at 340 ± 1 nm while the fluorescence spectrum peaks at 465 nm

Summary

Introduction

Cellular processes rely on energy produced during metabolism and a number of methodologies have been developed for its characterization. Nicotinamide Adenine Dinucleotide (NAD), a key coenzyme in metabolic pathways is frequently used as a metabolic fingerprint [1,2,3] This soluble compound is found in its free and bound form in the cytosol and mitochondria, respectively [4, 5]. Its fluorescence characteristics is virtually identical with that of nicotinamide adenine dinucleotide phosphate, (NADPH) [6] These two compounds are jointly referred to as NAD(P)H. Fluorescence lifetime imaging (FLIM) of endogenous cell fluorophores including NAD(P)H has been used to characterise the metabolic activity of cells and it has been related to free and bound NAD(P)H concentrations in cells [10, 11]. Our method uses low constant light irradiance and it does not require expensive systems

Methods

Results

Conclusion