Autofluorescence lifetime imaging resolves cell heterogeneity within peripheral blood mononuclear cells.

Significance: Standard methods to characterize peripheral blood mononuclear cells (PBMCs) are often destructive, lack metabolic information, or do not provide single-cell resolution. Label-free tools that nondestructively measure single-cell metabolism within PBMCs can provide new layers of information to characterize disease state and cell therapy potential. Aim: We aim to determine whether nondestructive fluorescence lifetime imaging microscopy (FLIM) of the endogenous metabolic cofactors nicotinamide adenine dinucleotide (phosphate) (reduced form) and flavin adenine dinucleotide (oxidized form), or optical metabolic imaging (OMI), can identify immune cell subsets and activation state within heterogeneous PBMC cultures. Approach: OMI measured single-cell metabolism of PBMCs from three different human donors in the quiescent or activated (phorbol 12-myristate 13-acetate and ionomycin) state. Fluorescent antibodies were used as ground truth labels for single-cell classifiers of immune cell subtypes. Results: OMI identified quiescent versus activated PBMCs with 94% accuracy at only 2 h post-stimulation, identified monocytes within quiescent and activated PBMCs with 96% and 88% recall, respectively, and identified NK cells within quiescent and activated PBMCs with 74% recall. Conclusion: OMI identifies activation state and immune cell subpopulations within PBMCs, enabling single-cell and label-free measurements of metabolic heterogeneity within complex PBMC samples. Therefore, OMI could enhance PBMC immunophenotyping for diagnostic and therapeutic applications.