Autofluorescence lifetime imaging resolves cell heterogeneity within peripheral blood mononuclear cells

Area of Science:

• Immunology
• Biotechnology
• Medical Diagnostics

Background:

• Standard peripheral blood mononuclear cell (PBMC) characterization methods are often destructive, lack metabolic insights, or fail to provide single-cell resolution.
• Label-free, non-destructive tools are needed to measure single-cell metabolism in PBMCs, offering new data for disease state and cell therapy assessment.

Purpose of the Study:

• To determine if non-destructive fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD (Optical Metabolic Imaging - OMI) can identify immune cell subsets and activation states within heterogeneous PBMC cultures.

Main Methods:

• Single-cell metabolism of PBMCs from three human donors was measured using OMI in quiescent and activated states.
• Phormid 12-myristate 13-acetate and ionomycin were used for cell activation.
• Fluorescent antibodies served as ground truth for single-cell immune classifier validation.

Main Results:

• OMI accurately identified quiescent versus activated PBMCs (93% accuracy) two hours post-stimulation.
• Monocyte identification accuracy was 96% in quiescent and 88% in activated PBMCs.
• NK cell identification accuracy was 74% in both quiescent and activated PBMCs.

Conclusions:

• OMI enables label-free, single-cell metabolic measurements within complex PBMC samples, identifying activation states and immune cell subpopulations.
• This technique can enhance PBMC immunophenotyping for diagnostic and therapeutic applications.
• Autofluorescence lifetime imaging offers a novel method to characterize immune cell metabolic activity, potentially improving disease diagnostics and cell therapy production.