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. 2018 Feb 2;8:13. doi: 10.3389/fonc.2018.00013

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Copyright © 2018 Sant’Anna-Silva, Santos, Campos, Oliveira Gomes, Pérez-Valencia and Rumjanek.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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NADH fluorescence lifetime distribution in ZsG, LN1, and LN2 cells. (A) Two-photon fluorescence microscopy intensity images showing NADH fluorescence of ZsG, LN1, and LN2 cells excited at 740 nm. (B) FLIM map of ZsG, LN1, and LN2 represented by a color-coded scale, by which pixels with a high free/bound NADH ratio are in purple, and pixels with a low free/bound NADH ratio are in red. All the lifetimes ratios are related to a specific color of the scale. (C) Phasor fingerprint, representing the mean S and G position of each image phasor distribution. (D) Mean S (left) and mean G (right) position of each cell line lifetimes distribution on phasor plot. Values represent mean ± SD; N = 5, with five images each; ***P < 0.0001. All the images were obtained on a two-photon microscope (Nikon Eclipse TE2000-U) and Plan-Apochromat 60×/1.20 water immersion objective lens (Nikon). Data were acquired on VistaVision software and Alba FLIM instrument (ISS) and analyzed on SimFCS software.