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. 2022 Oct 18;11:e77373. doi: 10.7554/eLife.77373

Figure 3. Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) metabolimaging analysis.

(A) Time-course imaging of representative (same field of view throughout) IFNγ-M1 macrophages, scale bar: 100 μm. (B, C) Average fluorescence lifetime (τavg) and optical redox ratio (ORR) values for IFNγ-M1 and IL-4-M2 when treated sequentially with oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone + antimycin A and 2-deoxy-d-glucose (2-DG) of a representative donor. (D) z-score heatmap of 2P-FLIM acquired data for six donors separated by macrophage polarisation and metabolic inhibitor, each individual row corresponds to an imaging field. (E) Uniform Manifold Approximate and Projection (UMAP) plot of 2P-FLIM variables after each treatment each dot corresponds to an individual imaging field. (F) UMAP plot of 2P-FLIM variables after FCCP treatment, each dot corresponds to an individual imaging field. (G) Receiver-operator curve and area under curve values of random forests machine learning model applied to 2P-FLIM data after FCCP treatment. (H) 2P-FLIM weight features determined by mean decrease accuracy and mean decrease Gini of random forests model used to classify macrophages.

Figure 3—source data 1. Fluorescence lifetime imaging microscopy (FLIM) imaging and corresponding variables measurement for each replicate, Uniform Manifold Approximate and Projection (UMAP) analysis, and machine learning (random forests) input data and coding.

Figure 3.

Figure 3—figure supplement 1. Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of IFNγ-M1 macrophages for basal conditions, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone with antimycin A, and 2-deoxy-d-glucose treatments with the concentrations detailed in the article.

Figure 3—figure supplement 1.

All treatment images collected at the 30 min of treatment and color-coded for different NADH FLIM variables.
Figure 3—figure supplement 2. Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of IL-4-M2 macrophages for basal conditions, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone with antimycin A, and 2-deoxy-d-glucose treatments with the concentrations detailed in the article.

Figure 3—figure supplement 2.

All treatment images collected at the 30 min of treatment and color-coded for different NADH FLIM variables.
Figure 3—figure supplement 3. Phasor analysis of NADH fluorescence lifetime imaging microscopy (FLIM) variables in basal and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) conditions.

Figure 3—figure supplement 3.

(A) Basal phasor with values set at 3.4 ns (red) and 0.4 ns (green) and all donors basal FLIM data distribution. (B) Histogram of all donors normalised to pixel intensity in basal conditions. (C) Histogram distribution of a representative donor normalised to pixel intensity in basal conditions. (D) FCCP phasor with values set at 3.4 ns (red) and 0.4 ns (green) and all donors FCCP FLIM data distribution. (E) Histogram of all donors (N = 6) normalised to pixel intensity in FCCP conditions. (F) Histogram distribution of a representative donor normalised to pixel intensity in FCCP conditions. Ψ value is the non-overlapped area for both curves in each plot when compared with the total area occupied by both curves. A higher Ψ value determines a better segregation between datasets.
Figure 3—figure supplement 3—source data 1. Phasor fluorescence lifetime imaging microscopy (FLIM) analysis raw histogram data.
Figure 3—figure supplement 4. 2P-FLIM variables correlation matrix.

Figure 3—figure supplement 4.

Principal component analysis (PCA) and t-SNE data visualisation of two-photon fluorescence lifetime imaging microscopy (2P-FLIM) variables obtained from full field of view (FoV) collected during the course of metabolic treatments (A, B). PCA data visualisation of 2P-FLIM variables obtained from full FoV collected during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) treatments and representation of 99.9% confidence ellipses derived from 3 standard deviations of the data presented for each group of samples (C). t-SNE data visualisation of 2P-FLIM variables obtained from full FoV collected during FCCP treatments (D).
Figure 3—figure supplement 5. Correlation matrix of two-photon fluorescence lifetime imaging microscopy (2P-FLIM) NAD(P)H variables with correlation values displayed.

Figure 3—figure supplement 5.

Figure 3—figure supplement 6. Dispersion plot distribution of photons/pixel per fluorescence lifetime variable value.

Figure 3—figure supplement 6.

(A) τ1, (B) τ2, (C) α1, (D) α2, (E) optical redox ratio (ORR) distribution based on NADH results, (F) ORR distribution based on FAD results, and (G) τavg. These values are obtained after background removal.
Figure 3—figure supplement 6—source data 1. Dispersion plot distribution of photons/pixel per fluorescence lifetime variable value raw data.