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

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.

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

“…Therefore the possibility of evaluating the fractional contributions of bound and unbound NAD(P)H (i.e., S/L ratio) (74,75) is possible using the phasor analysis. Often, endogenous fluorophores exhibit two or more exponential decay profiles owing to the distribution of bound and free NAD(P)H inside the cell.…”

“…Often, endogenous fluorophores exhibit two or more exponential decay profiles owing to the distribution of bound and free NAD(P)H inside the cell. Therefore the possibility of evaluating the fractional contributions of bound and unbound NAD(P)H (i.e., S/L ratio) (74,75) is possible using the phasor analysis. Phasor graphical analyses that are derived from FLIM data utilize the reciprocal property of phasors; "gated" pixels yield a lifetime image map of a handful of cells providing an evaluation of the multiple decay kinetics.…”

A deeper understanding of intestinal organoid metabolism revealed by combining fluorescence lifetime imaging microscopy (FLIM) and extracellular flux analyses

“…Without considering potential problems of probe delivery and calibration performance 45 , most tested phosphorescent probes provide mono-exponential decay fits, welldocumented physical interactions with O2, ensuring a high precision of measurements and data interpretation 42,47 . In contrast, most fluorescent dyes, probes and fluorescent protein biosensors display double or multi-exponential decay fits, which are meaningful but require complex interpretation or the use of phasor approach plotting 27,32,48 . Thus, detailed interpretation of NAD(P)H imaging is not straightforward, often demanding additional pharmacological tests, segmentation based on signal localization and potentially use of additional tracers 27,28,32,44 .…”

Non-Euclidean phasor analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy

“…The separation of fluorescent species based on their lifetime is best achieved using the phasor approach. 18,[27][28][29] This method is derived from Fourier analysis, where rather than fitting a linear combination of decaying exponentials onto a decay trace I ij ðtÞ in the temporal domain, the data are transformed into a phasor with coordinates ðG ij ; S ij Þ defined as E Q -T A R G E T ; t e m p : i n t r a l i n k -; e 0 0 1 ; 3 2 6 ; 6 6 8…”