Abstract
Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.
Highlights
The demand for methods evaluating adipose tissue metabolism is growing rapidly, since obesity related diseases such as hypertension, type 2 diabetes, and many forms of cancer are increasing drastically[1,2]
This study shows, for the first time, that four fluorescence lifetime components exist for NADH in adipocytes, and that lifetime pairs can be localized to distinct cellular compartments
The fluorescence lifetime component C1 (0.5 ns) and C3 (2.3 ns) are predominantly found in mitochondria, while the lifetime components C2 (1 ns) and C4 (3.7 ns) predominantly arise from the cytoplasm. This is in contrast to the traditional two lifetime model for NADH, where the short fluorescence lifetime τ1 (0.5 ns) represents the cytoplasm and the long fluorescence lifetime τ2 (2.5 ns) is attributed to the mitochondria[25]
Summary
The demand for methods evaluating adipose tissue metabolism is growing rapidly, since obesity related diseases such as hypertension, type 2 diabetes, and many forms of cancer are increasing drastically[1,2]. While traditional assessment of NADH in cell cultures and tissue only shows two fluorescence lifetimes components, one free and one bound, recent studies in cancer cells and cerebral tissue suggest that there are likely four distinct NADH species[16,24]. Mapping the four lifetime components to specific cellular components quantitatively reflects changes in mitochondrial and cytoplasmic NADH concentrations and resulted in superior metabolic assessment compared to traditional FLIM analysis. It was evaluated whether changes in fluorescence lifetime correlate with gene expression of BAT-related markers such as UCP1. By understanding how fluorescence lifetime can be related to metabolic activity, this study constitutes a powerful imaging toolkit for cellular metabolism investigations and a valuable tool in obesity research
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