Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level

Abstract

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed noninvasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ~ 1.8 fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo

“…As described previously [19], data were processed using custom designed software developed in Matlab and employing metrics derived from FLIM data as detailed in Vishwasrao et al and Yu et al [13,14]. To characterize the system's instrument response function (IRF) and to aid with data processing, FLIM measurements were recorded of dissolved NADH (0.5 mM in saline) at the beginning or conclusion of each experiment.…”

“…Time-resolved decay profiles of NADH autofluorescence permit distinction of multiple fluorescence lifetime components associated with different 'NADH species,' which reportedly reflect differences in NADH enzymatic binding. Investigators have demonstrated variations in FLIM-based observations of NADH in response to pharmacological and physiological disruptions and disease progression in cell cultures, tissue slices, and in the liver in vivo [13][14][15][16][17][18]. We previously demonstrated in vivo 2PM-based FLIM (2P-FLIM) measurements of cerebral NADH in anesthetized rats, and observed how cerebral NADH fluorescence can be resolved into 4 distinct lifetime components whose amplitudes change rapidly with anoxia and recovery [19].…”

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

“…Because excitation in the 350-450-nm wavelength range is highly toxic to cells, and because shifts in the emission spectra due to molecular interactions in cells make the analysis difficult, this approach of metabolic mapping in living cells and tissues became obsolete. However, the application of dual-photon excitation of NADH at 730 nm, which reduces cytotoxicity considerably, the determination of the ratio of free and protein (enzyme)-bound NADH using fluorescence lifetime imaging microscopy and anisotropy of autofluorescence, and the application of fast photon detection re-introduced NADH as an attractive probe for spatially resolved imaging of the metabolism of living cells (Ramanujam et al 1994;Bird et al 2005;Ramanujan et al 2008;Yu and Heikal 2009). The focus of these studies is particularly on metabolic mapping of normal and malignant cells and tissues.…”

Frex and FrexH

“…The ratio of free/bound NADH varies significantly from 1.5:1 20 to 1:4. 21 For example, Wakita et al 23 were unable to detect free NADH in rat liver mitochondria regardless of their respiratory state, whereas Blinova et al 20 observed a high proportion of free NADH in pig heart mitochondria using a similar technique. Interestingly, Kasimova et al suggested that the free NADH concentration in plant mitochondria is kept constant under different metabolic conditions.…”

“…15 The mitochondrial NAD + -NADH ratio also varies from 2 to 16 in different reports. [16][17][18] In recent years, an increasing number of studies on free NADH were largely focused on measurements via time-resolved fluorescence, 19,20 fluorescence anisotropy, 21,22 and fluorescence spectral decomposition analysis. 16 These techniques enable researchers to distinguish between the protein-bound and free NADH in the intracellular environment.…”