Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM

Horst Wallrabe,Ammasi Periasamy,Shagufta R Alam,Zdenek Svindrych,Karsten H Siller,Song Hu,David Kashatus,Tianxiong Wang

doi:10.1038/s41598-017-18634-x

Horst Wallrabe, Ammasi Periasamy + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-18634-x

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Abstract

Multiphoton FLIM microscopy offers many opportunities to investigate processes in live cells, tissue and animal model systems. For redox measurements, FLIM data is mostly published by cell mean values and intensity-based redox ratios. Our method is based entirely on FLIM parameters generated by 3-detector time domain microscopy capturing autofluorescent signals of NAD(P)H, FAD and novel FLIM-FRET application of Tryptophan and NAD(P)H-a2%/FAD-a1% redox ratio. Furthermore, image data is analyzed in segmented cells thresholded by 2 × 2 pixel Regions of Interest (ROIs) to separate mitochondrial oxidative phosphorylation from cytosolic glycolysis in a prostate cancer cell line. Hundreds of data points allow demonstration of heterogeneity in response to intervention, identity of cell responders to treatment, creating thereby different sub-populations. Histograms and bar charts visualize differences between cells, analyzing whole cell versus mitochondrial morphology data, all based on discrete ROIs. This assay method allows to detect subtle differences in cellular and tissue responses, suggesting an advancement over means-based analyses.

Highlights

Applications of Fluorescence Lifetime Imaging Microscopy (FLIM) have grown exponentially in a broad range of life-sciences and industrial fields, a reflection of specific advantages over intensity-based microscopy[1,2,3,4,5]
The coenzymes NADH and FAD are involved in catabolic reactions of amino acid and fatty acid oxidation, glycolysis, citric acid cycle and in electron transport chain (ETC) which results in energy generation by oxidative phosphorylation (OXPHOS)
A higher glycolytic rate in cancer is a less efficient way of producing energy (2Pyruvate + 2ATP + 2NADH) than the low glycolytic rate and mitochondrial oxidation of pyruvate (36 ATP) seen in normal cells[18]. Cancer cells shift their metabolism to the production of lactate from pyruvate in the cytosol by the enzyme Lactate dehydrogenase (LDH), in the process oxidizing the NADH and regenerating NAD+ required for ATP production through glycolysis

Summary

Introduction

Applications of Fluorescence Lifetime Imaging Microscopy (FLIM) have grown exponentially in a broad range of life-sciences and industrial fields, a reflection of specific advantages over intensity-based microscopy[1,2,3,4,5]. Fluorescence lifetime is independent of fluorophore concentration, which makes it a valuable tool for quantitative studies in scattering and absorbing samples. Both frequency domain and time domain FLIM methods have been applied[14,15,16]. Mitochondrial oxidative phosphorylation (OXPHOS) activity consumes NADH (increased NADH-enzyme-bound fraction) and produces FAD (diminished FAD enzyme-bound fraction). Both the co-enzymes in their reduced (NAD(P)H and FADH2) and oxidized (NAD(P)+ and FAD) forms participate in the cellular oxidation-reduction reactions critical for cell physiology. Mitochondria, the power house of a cell has prominent and discrete signals from NAD(P)H and FAD and provides a “consumer report” of energy expenditure and generation, its redox state and the level of metabolic activity[29]

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