NSOM/QD-based fluorescence–topographic image fusion directly reveals nano-spatial peak–valley polarities of CD69 and CD71 activation molecules on cell-membrane fluctuations during T-cell activation

Nano-spatial distribution of cell surface molecules on cell membrane fluctuations during T-cell activation has not been reported. In this study, we innovated application of near-field scanning optical microscopy (NSOM)/quantum dots (QDs)-based nanotechnology through three-dimensional image fusion algorithm to merge the simultaneously obtained dual-color fluorescence information and three-dimensional topography. This novel imaging system made it possible to visualize nano-spatial distribution and organization of early-activation molecules CD69 and late-activation molecules CD71 on cell-membrane fluctuations during T-cell activation. Interestingly, most CD69 molecules were clustered to form 250–500nm nano-domains polarizing predominantly in the peak of the cell-membrane fluctuations. In contrast, although CD71 molecules were also clustered as 250–500nm nano-domains, they polarized dominantly in the valley of the cell-membrane fluctuations. The peak–valley polarities of CD69 nano-domains and CD71 nano-domains implied their different functions. CD69 nano-domains polarizing on membrane-peak fluctuations might serve as transient platforms driving TCR/CD3-induced signaling and activation, whereas CD71 nano-domains distributing in the membrane-valley fluctuations appeared to facilitate iron uptake for increased metabolisms in T-cell activation. Importantly, this NSOM/QD-based fluorescence–topographic image fusion provides a powerful tool to visualize nano-spatial distribution of cell-surface molecules on cell-membrane fluctuations and enable better understanding of distribution–function relationship.