Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy.

The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet.