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

View Article: PubMed Central - PubMed

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.

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FLIM characteristics of 3D Caco-2 luminal cysts.(a) EGF induces a significant decrease in the short fluorescence lifetime τ1 of NADH (n = 4, *p = 0.002). (b) EGF treatment induces a significant decrease in τ2 (n = 4, *p = 0.0391). (c) Treatment of 3D Caco-2 models with EGF induces an increase in the short fluorescence lifetime contribution α1MEDF (n = 4, *p = 0.0213). (d,e) False color-coded images of (d) untreated and (e) EGF-treated 3D Caco-2 luminal cyst models indicating the range of α1MEDF from 65% (red) to 80% (blue).
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f3: FLIM characteristics of 3D Caco-2 luminal cysts.(a) EGF induces a significant decrease in the short fluorescence lifetime τ1 of NADH (n = 4, *p = 0.002). (b) EGF treatment induces a significant decrease in τ2 (n = 4, *p = 0.0391). (c) Treatment of 3D Caco-2 models with EGF induces an increase in the short fluorescence lifetime contribution α1MEDF (n = 4, *p = 0.0213). (d,e) False color-coded images of (d) untreated and (e) EGF-treated 3D Caco-2 luminal cyst models indicating the range of α1MEDF from 65% (red) to 80% (blue).

Mentions: 3D Caco-2 luminal cysts left untreated or treated with EGF were imaged using FLIM. A bi-exponential decay fitting was applied to the obtained fluorescence decay profiles of the models and from this, the individual contributing lifetime components of free NADH (τ1), protein-bound NADH (τ2), and the contribution of their lifetimes to the overall signal (α1MEDF being the contribution of τ1, Fig. 3) were determined. The treatment of EGF had a statistically significant effect on τ2 and α1MEDF in the 3D Caco-2 luminal cyst models (Fig. 3a–c). Specifically, EGF induced a significantly higher value for τ2 (2853 ± 75 ps versus 3000 ± 4 ps, n = 4, p=0.0261, Fig. 3b), and a significantly increased value for α1MEDF (72.28 ± 0.52% versus 69.83 ± 0.62%, n = 4, p = 0.002, Fig. 3c). No statistically significant difference in τ1 was detected when comparing models treated with EGF with those left untreated. The results from the multi-exponential decay fitting can be viewed qualitatively in the false colour-coded micrographs provided in Fig. 3e,f. These micrographs depict the value and change of α1MEDF of untreated 3D Caco-2 luminal cyst models with and without EGF treatment.


Applying phasor approach analysis of multiphoton FLIM measurements to probe the metabolic activity of three-dimensional in vitro cell culture models
FLIM characteristics of 3D Caco-2 luminal cysts.(a) EGF induces a significant decrease in the short fluorescence lifetime τ1 of NADH (n = 4, *p = 0.002). (b) EGF treatment induces a significant decrease in τ2 (n = 4, *p = 0.0391). (c) Treatment of 3D Caco-2 models with EGF induces an increase in the short fluorescence lifetime contribution α1MEDF (n = 4, *p = 0.0213). (d,e) False color-coded images of (d) untreated and (e) EGF-treated 3D Caco-2 luminal cyst models indicating the range of α1MEDF from 65% (red) to 80% (blue).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304149&req=5

f3: FLIM characteristics of 3D Caco-2 luminal cysts.(a) EGF induces a significant decrease in the short fluorescence lifetime τ1 of NADH (n = 4, *p = 0.002). (b) EGF treatment induces a significant decrease in τ2 (n = 4, *p = 0.0391). (c) Treatment of 3D Caco-2 models with EGF induces an increase in the short fluorescence lifetime contribution α1MEDF (n = 4, *p = 0.0213). (d,e) False color-coded images of (d) untreated and (e) EGF-treated 3D Caco-2 luminal cyst models indicating the range of α1MEDF from 65% (red) to 80% (blue).
Mentions: 3D Caco-2 luminal cysts left untreated or treated with EGF were imaged using FLIM. A bi-exponential decay fitting was applied to the obtained fluorescence decay profiles of the models and from this, the individual contributing lifetime components of free NADH (τ1), protein-bound NADH (τ2), and the contribution of their lifetimes to the overall signal (α1MEDF being the contribution of τ1, Fig. 3) were determined. The treatment of EGF had a statistically significant effect on τ2 and α1MEDF in the 3D Caco-2 luminal cyst models (Fig. 3a–c). Specifically, EGF induced a significantly higher value for τ2 (2853 ± 75 ps versus 3000 ± 4 ps, n = 4, p=0.0261, Fig. 3b), and a significantly increased value for α1MEDF (72.28 ± 0.52% versus 69.83 ± 0.62%, n = 4, p = 0.002, Fig. 3c). No statistically significant difference in τ1 was detected when comparing models treated with EGF with those left untreated. The results from the multi-exponential decay fitting can be viewed qualitatively in the false colour-coded micrographs provided in Fig. 3e,f. These micrographs depict the value and change of α1MEDF of untreated 3D Caco-2 luminal cyst models with and without EGF treatment.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus