Label-free optical metabolic imaging of adipose tissues for prediabetes diagnosis

Figures

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  Figure 1. Lipofuscin-like fluorescence can be used as a label-free marker for adipose tissue macrophages. (A) Co-localization of lipofuscin-like red autofluorescence and macrophage marker F4/80 in the epididymal fat of C57 mice. FITC-anti-mouse F4/80 excited by 960 nm was presented by green color, and lipofuscin-like red autofluorescence (Red FL) excited by 1040 nm was presented by red color. Scale bar: 50 μm. (B) (left panel) Intensity profiles of FITC and lipofuscin-like red autofluorescence within the red rectangular region of the merged image (inset). (right panel) PCC = 0.57 ± 0.052, MCC-M1= 0.82 ± 0.045, and MCC-M2 = 0.65 ± 0.053. (C) Co-localization of lipofuscin-like red autofluorescence and GFP in the epididymal fat of C57BL/6J-c2J-LysM-eGFP mice. Scale bar: 50 μm. (D) (left panel) Intensity profiles of GFP and lipofuscin-like fluorescence within the red rectangular region of the merged image (inset). (right panel) PCC = 0.69 ± 0.058, MCC-M1 = 0.97 ± 0.008, MCC-M2 = 0.94 ± 0.01. (E) Co-localization of lipofuscin-like red autofluorescence and lysosome in the epididymal fat of C57 mice. Lysosome was identified with lysotracker green. Scale bar: 20 μm. (F) (left panel) Intensity…

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  Figure 2. Optical readouts of adipocytes and macrophages in response to the prediabetic microenvironment of epididymal adipose tissue. (A) Changes of HOMA-IR index in mice fed HFD or HFHSD for 16 weeks. The red dotted line marks the development of IR in mice fed HFD or HFHSD for 5 weeks. (B) Fasting glucose levels in mice fed HFD or HFHSD for 16 weeks. The red dotted line marks the blood glucose higher than 13mmol/L in mice fed HFD or HFHSD for 16 weeks. (C) Representative images of NAD(P)H, FAD, and lipofuscin-like fluorescence intensity in epididymal fat from mice fed HFD or HFHSD for 1 month. Scale bar: 50 μm. (D) FAD/[NAD(P)H + FAD] intensity of adipocyte cytoplasm was quantified. (E) Quantification of redox ratio of adipose tissue macrophages. (F) Pseudo-color-coded FLIM a1 and t2 images of NAD(P)H in adipose tissues. Scale bar: 50 μm. (G) The a1-t2 scatter plot of adipocytes. Control: orange, HFD: olive, and HFHSD: royal. (H) The a1-t2 scatter plot of macrophages. (I, J) ROC curves and AUC values for optical readouts (redox ratio, a1, t1, t2, and t2-a1-redox ratio-integrated parameter) of adipocytes, showing their ability to distinguish between (I) control and HFD groups or between…

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  Figure 3. TPFM and FLIM metabolic imaging of epididymal adipose tissues of diabetic mice. (A) Representative images of NAD(P)H, FAD, and lipofuscin-like fluorescence intensity in epididymal adipose tissues from mice fed HFD or HFHSD for 4 months. Scale bar: 50 μm. (B) Quantification of macrophages' redox ratio. (C) The redox ratio of macrophages in CLSs and non-CLSs (Left: HFD group; right: HFHSD group) was quantified. (D) Pseudo-color-coded FLIM a1 and t2 images of NAD(P)H in adipose tissues. Scale bar: 50 μm. (E) The a1-t2 scatter plot of macrophages. (F) Quantification of the relative fluorescence intensity of lipofuscin-like pigments in macrophages. (G, H) ROC curves and AUC values for optical readouts (redox ratio, a1, t1, t2, lipofuscin intensity, and t2- t1-a1-redox ratio-lipofuscin-integrated parameter) of macrophages, showing their ability to distinguish between (G) control and HFD groups or between (H) control and HFHSD groups. (I) Heatmap of OXPHOS-related and inflammation-related genes expression in adipose tissues of normal and diabetic mice. Data in (B, C, F) are presented as Mean ± SD (n = 8). **P < 0.01 and ***P < 0.001 relative to the control group. Each group in (B, C, E,…

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  Figure 4. Integrated analysis of metabolic imaging readouts enables the differential diagnosis of diabetes stages. (A, B) Z-score heatmaps showing changes in optical readouts of adipocytes and macrophages in healthy control, prediabetes, and diabetes conditions. (C, D) PCA scores plots (PC1 versus PC2) for the differentiation of integrated metabolic imaging readouts from control, prediabetic (HFD and HFHSD), and diabetic (HFD and HFHSD) mice. For each group, 25-30 data points were obtained from 8 biological replicas. (E, F) ROC curves based on the optical metrics of adipose tissues for differential diagnosis of prediabetes versus diabetes, control versus prediabetes, and control versus diabetes.

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  Figure 5. Remission of IR in mice restoring the redox ratio and NAD(P)H lifetime of adipose tissue to normal levels. (A) Representative images of NAD(P)H, FAD, and lipofuscin fluorescence intensity. Scale bar: 50 μm. Relative FAD/[NAD(P)H + FAD] intensities in adipocyte cytoplasm (B) and macrophages (C) in adipose tissues were quantified. (D) False color-coded FLIM a1 and t2 images of NAD(P)H in adipose tissues. Scale bar: 50 μm. (E) The a1-t2 scatter plot showed that no difference in NAD(P)H lifetime of adipocytes was observed among the three groups. Data are shown as Mean ± SD (n = 8). Each group in (B, C, and E) contains 25-30 data points from 8 biological replicas. Each data point is the average of 10 cells in one field of view.