Systematic Enzyme Mapping of Cellular Metabolism by Phasor-Analyzed Label-Free NAD(P)H Fluorescence Lifetime Imaging

Abstract

In the past years, cellular metabolism of the immune system experienced a revival, as it has become clear that it is not merely responsible for the cellular energy supply, but also impacts on many signaling pathways and, thus, on diverse cellular functions. Label-free fluorescence lifetime imaging of the ubiquitous coenzymes NADH and NADPH (NAD(P)H-FLIM) makes it possible to monitor cellular metabolism in living cells and tissues and has already been applied to study metabolic changes both under physiologic and pathologic conditions. However, due to the complex distribution of NAD(P)H-dependent enzymes in cells, whose distribution continuously changes over time, a thorough interpretation of NAD(P)H-FLIM results, in particular, resolving the contribution of various enzymes to the overall metabolic activity, remains challenging. We developed a systematic framework based on angle similarities of the phase vectors and their length to analyze NAD(P)H-FLIM data of cells and tissues based on a generally valid reference system of highly abundant NAD(P)H-dependent enzymes in cells. By using our analysis framework, we retrieve information not only about the overall metabolic activity, i.e., the fraction of free to enzyme-bound NAD(P)H, but also identified the enzymes predominantly active within the sample at a certain time point with subcellular resolution. We verified the performance of the approach by applying NAD(P)H-FLIM on a stromal-like cell line and identified a different group of enzymes that were active in the cell nuclei as compared to the cytoplasm. As the systematic phasor-based analysis framework of label-free NAD(P)H-FLIM can be applied both in vitro and in vivo, it retains the unique power to enable dynamic enzyme-based metabolic investigations, at subcellular resolution, in genuine environments.

Discriminating different grades of cervical intraepithelial neoplasia based on label-free phasor fluorescence lifetime imaging microscopy

“…Fluorescence lifetime imaging microscopy (FLIM) can be used to monitor cellular metabolic abnormalities, tissue lesions, and the rates of glycolysis vs. oxidative phosphorylation by detecting lifetime changes in NAD(P)H and FAD in cells or tissues [19][20][21][22][23][24]. The metabolism is maintained in an abnormal state throughout the entire process from normal to low-risk lesions and then high-risk lesions.…”

Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages

“…Importantly, changes in mitochondrial membrane potential and/or the integrity of the ER also lead to varying calcium concentrations within the cytoplasm since both act as major intracellular calcium buffering organelles ( Kass and Orrenius, 1999 ) A close connection between mitochondrial calcium homeostasis, altered reactive oxygen speciesproduction, and the expression of plasma cell master transcription factor BLIMP1, as well as changes in metabolism, has been reported previously ( Jang et al, 2015 ; Shanmugapriya et al, 2019 ). We have recently applied phasor-FLIM of endogenous NAD(P)H fluorescence for mapping of metabolic enzyme activities in cell cultures ( Leben et al, 2019 ). The combination of this technique with FLIM-based intravital calcium analysis will help to further dissect immunometabolic processes in B cells, as well as in short-lived plasma cells and LLPCs in vivo.…”

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

“…In order to characterize metabolic conditions in the tissue on a cellular level, related to its perfusion, and analyze their impact on bone regeneration , the combination of Limbostomy with fluorescence lifetime imaging (FLIM) holds great potential. FLIM allows the marker‐free investigation of the NAD(P)H‐related metabolism of cells . To overcome the limitation of decreased imaging depth in mineralized tissue, three‐photon microscopy could be an interesting technique that may also be used in combination with Limbostomy.…”