T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium ([Ca2+]iand leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the contact requirements for T-cell activation, using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent [Ca2+]iresponse, detected as an emission shift from the combination of fura-red and oregon-green, two cytoplasmic [Ca2+] indicators. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling (2-5 μm/min). T cells contacted with antigen-presenting cells or antibody-coated beads entered a dynamic and reproducible program in the first 10-20 mins, including [Ca2+]iincrease, changes in shape and motility, engulfment, and stable contact. T cells presented with antigen at the leading edge had a higher probability of responding (85%) and a shorter latency of response (50 secs) than those contacting APCs or beads with their trailing end (APCs: 30%, 150 secs; beads: 6%, 300 secs). Alterations in antibody density, quantified by FACS analysis, and bead size were used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Preliminary data show that T cell responses (response percentage, latency and [Ca2+]ipattern) depend on both antibody density and bead size.