Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models

Pirmin H Lakner,Katja Schenke-Layland,Monilola A Olayioye,Michael G Monaghan,Yvonne Möller

doi:10.1038/srep42730

Abstract

Fluorescence lifetime imaging microscopy (FLIM) can measure and discriminate endogenous fluorophores present in biological samples. This study seeks to identify FLIM as a suitable method to non-invasively detect a shift in cellular metabolic activity towards glycolysis or oxidative phosphorylation in 3D Caco-2 models of colorectal carcinoma. These models were treated with potassium cyanide or hydrogen peroxide as controls, and epidermal growth factor (EGF) as a physiologically-relevant influencer of cell metabolic behaviour. Autofluorescence, attributed to nicotinamide adenine dinucleotide (NADH), was induced by two-photon laser excitation and its lifetime decay was analysed using a standard multi-exponential decay approach and also a novel custom-written code for phasor-based analysis. While both methods enabled detection of a statistically significant shift of metabolic activity towards glycolysis using potassium cyanide, and oxidative phosphorylation using hydrogen peroxide, employing the phasor approach required fewer initial assumptions to quantify the lifetimes of contributing fluorophores. 3D Caco-2 models treated with EGF had increased glucose consumption, production of lactate, and presence of ATP. FLIM analyses of these cultures revealed a significant shift in the contribution of protein-bound NADH towards free NADH, indicating increased glycolysis-mediated metabolic activity. This data demonstrate that FLIM is suitable to interpret metabolic changes in 3D in vitro models.

Highlights

Fluorescence lifetime imaging microscopy (FLIM) is a useful imaging tool that can be utilized for the non-invasive characterisation of biological samples in vitro[1] and in vivo[2], and serve as a method for in situ diagnosis of pathological tissues[3]
An instrument response function (IRF) data file was recorded from urea crystals and applied to minimize the chi-squared fit χ2, which is an indicator of how the measured data and fitted equations are related. χ2 = 1 represents complete unity between the measured data and theoretical fit. 3D Caco-2 luminal cysts were imaged using FLIM before and after the application of KCN or H2O2
Intracellular NADH is a universal cofactor that is involved in many metabolic reactions, especially in cell energy metabolism[32]

Summary

Introduction

Fluorescence lifetime imaging microscopy (FLIM) is a useful imaging tool that can be utilized for the non-invasive characterisation of biological samples in vitro[1] and in vivo[2], and serve as a method for in situ diagnosis of pathological tissues[3]. NADH can occur in a free or protein-bound state in cells, which is discriminated due to the fluorescence decay time of the emission signal that is measurable using FLIM. Where I(t) is the intensity of photons at time t and C is offset (e.g. background noise, room light, scattering effects), and the contributing lifetimes and their distributions are estimated using multiple iterations (please see the Supplementary data file for a more detailed explanation). Another approach to calculate fluorescence lifetimes is the phasor approach[14,15]. An exponential decay curve can be mathematically modelled using the following equation: exp −

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