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We have developed a novel method for activating T-cells on material surfaces that enable individual and population-based analyses of intracellular calcium flux, as a quantitative measure of T-cell receptor engagement. Functionalized material surfaces were created using a plasma-polymerized foundation layer to immobilize stimulatory T-cell ligands, which could induce T-cell receptor-dependent calcium flux in naive T-cells. Real-time confocal microscopic detection and quantification of calcium flux using paired fluorescent ratiometric probes facilitated the tracking and analysis of response profiles of individual T-cells, as well as population analyses using a combination of individual T-cell events. This type of combined analysis cannot be achieved using traditional population-based flow cytometric approaches, and thus provides a logical step towards developing the capacity to assess the magnitude and quality of inherently heterogeneous effector T-cell responses to antigenic challenge. Refereed/Peer-reviewed