Non-Invasive classification of macrophage polarisation by 2P-FLIM and machine learning

Abstract

In this study, fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence is applied as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages were obtained from human blood-circulating monocytes, polarised using established treatments, and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Fluorescence lifetime imaging microscopy (FLIM) quantified variations in NAD(P)H-derived fluorescent lifetimes in large field-of-view images of individual polarised macrophages also challenged, in real-time with small molecule perturbations of metabolism during imaging. We uncover FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) which strongly stratifies the phenotype of polarised human macrophages. This stratification and parameters emanating from a FLIM approach, served as the basis for machine learning models. Applying a random forest model, identified three strongly governing FLIM parameters, achieving a ROC AUC value of 0.944 when classifying human macrophages.

2P-FLIM unveils time-dependent metabolic shifts during osteogenic differentiation with a key role of lactate to fuel osteogenesis via glutaminolysis identified

“…After imaging, the sample can be still used for further metabolic, biochemical or histological validation [17]. In addition, it is possible to include machine learning models to the data analysis to further understand the impact of metabolic treatments [18].…”

“…After imaging, the sample can be still used for further metabolic, biochemical or histological validation [17]. In addition, it is possible to include machine learning models to the data analysis to further understand the impact of metabolic treatments [18]. hMSCs metabolic probing during differentiation using 2P-FLIM has been previously reported yet several of these studies are limited to direct observation of metabolic changes at a specific time-point without other biochemical validation such as evaluation of extracellular fluxes, measuring metabolites concentration or observing mitochondrial shape or biogenesis.…”