Protocol-optimized wide-field optical redox imaging for T cell activation and its potential for immunological research

SignificanceT cell activation and function are tightly linked to metabolic reprogramming, yet accessible, label-free methods to monitor these changes remain limited. Optical redox imaging (ORI) with two-photon microscopy has been utilized for studying metabolic functions and activation of immune cells, but this approach requires complex systems and is not as accessible and low-cost as single-photon wide-field fluorescence microscopy.AimWe aim to develop and validate wide-field ORI for assessing metabolic states and heterogeneity in human T cells.ApproachA wide-field inverted fluorescence microscope was used to perform ORI in vitro to evaluate and differentiate between unstimulated T cells and natural killer (NK) cells to demonstrate the technical feasibility of a wide-field system for studying immune cells. To avoid misrepresentation of activated T cells, an ORI protocol was optimized for imaging T cells from human donors seeded in and adherent to noncoated glass-bottom dishes. Intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and oxidized flavoproteins (Fp) was measured to compute the optical redox ratio (ORR= Fp/[NAD(P)H+Fp]). Quiescent and anti-CD3/CD28/CD2-activated T cells were characterized and compared, along with their responses to mitochondrial modulators of oxidative phosphorylation (OXPHOS).ResultsWide-field ORI differentiated total, CD4+, and CD8+ T cells from NK cells based on Fp, NAD(P)H, and ORR indices. Activation significantly increased NAD(P)H levels and decreased ORR in total, CD4+, and CD8+ T cells, indicating a more reduced mitochondrial redox state. ORI distinguished quiescent from activated T cells and revealed differences in metabolic heterogeneity and responses to OXPHOS perturbations. ORR images and histogram analysis of ORR indicated the existence of redox subpopulations in T cell samples.ConclusionsGenerating results consistent with ORI using two-photon microscopy, single-photon wide-field ORI provides a sensitive, low-cost, and nondestructive platform for characterizing immune cell metabolism and function. This approach with wide accessibility will broaden the applications of ORI for studying immune cell function and therapeutic responses.