Metabolic imaging using two-photon excited NADH intensity and fluorescence lifetime imaging

Abstract

Metabolism and mitochondrial dysfunction are known to be involved in many different disease states. We have employed two-photon fluorescence imaging of intrinsic mitochondrial reduced nicotinamide adenine dinucleotide (NADH) to quantify the metabolic state of several cultured cell lines, multicell tumor spheroids, and the intact mouse organ of Corti. Historically, fluorescence intensity has commonly been used as an indicator of the NADH concentration in cells and tissues. More recently, fluorescence lifetime imaging has revealed that changes in metabolism produce not only changes in fluorescence intensity, but also significant changes in the lifetimes and concentrations of free and enzyme-bound pools of NADH. Since NADH binding changes with metabolic state, this approach presents a new opportunity to track the cellular metabolic state.

Figure 1

Metabolic FLIM analysis procedure to characterize intracellular NADH pools in cyanide-inhibited Rat Basophilic Leukemia cells.. A) NADH fluorescence intensity B) Intensity-weighted lifetime image with each pixel fit to a single-exponential decay (Eq. 2). C) Intensity-weighted effective lifetime image with each pixel fit by a double-exponential decay (Eq. 3). The pseuodocolor scale for A) and B) is shown at right. D) Short and E) long lifetime image maps (scale bar lifetimes in ps) and relative NADH concentrations associated with the short and long lifetime components as calculated by Eq. 8.

Figure 2

NADH lifetime pixel histograms change with change in metabolic state. Sample FLIM analysis of RBL leukemia cells that was performed on A) normal, untreated cells, and metabolically B) inhibited, and C) uncoupled cells. Thin yellow lines show individual, Gaussian fits to the histogramed data, while the thick red line shows the sum of the individual fits. As shown in this figure, four Gaussians were sufficient to fit most datasets.

Figure 3

NADH lifetimes (A), subpopulation concentration distributions (B), and total concentration (C) vary with metabolic state in RBL cells. Because NADH pool concentrations shift with metabolic state, the percent reduction of NADH is significantly overestimated by intensity imaging alone (D).

Figure 4

NADH lifetimes (A) and subpopulation concentrations (B) vary with glucose concentration in adherent EMT6 Adenocarcinoma cells. While NADH fluorescence increased significantly (D), NADH concentration tends to decrease with increasing glucose concentration (C), indicating that the brightness increase was due to a change in the quantum yield of NADH.

Figure 5

A) Free NADH in solution is characterized by fluorescence decay times of 390 +/− 20 ps and 1140 +/− 60 ps. (B) Approximately 95% of the total NADH concentration has the shorter lifetime. (C) These characteristics are independent of the NADH concentration. (D) FLIM analysis shows a linear increase in concentration, as expected. These results can be understood by considering that free NADH in solution can fluctuate between an extended and a folded conformation (afterScott et al. (1970).

Figure 6

NADH lifetimes (A) and pool concentrations (B) are modified upon binding to Lactate Dehydrogenase. Four lifetimes are evident depending on the mole ratio of [NADH]/[LDH].