T cells play a pivotal role in the immune system's response to various conditions. They are activated by antigen-presenting cells (APCs) via T-cell surface receptors, resulting in cytokine production and T-cell proliferation. These interactions occur through the formation of immunological synapses. The advent of imaging flow cytometry has enabled detailed statistical analyses of these cellular interactions. However, the dynamics of T-cell receptors in response to in vitro stimulation are yet to receive attention, despite it being a crucial aspect of understanding T-cell behavior. In this article, we explore the responses of T cells to in vitro antibody-based stimulation without APCs. Specifically, we established a Th1 cell clone, subjected it to a combination of centrifugation-induced mechanical stress and anti-human CD3 and anti-human CD28 antibody stimulation as the in vitro antibody-based stimulation, and captured and analyzed bright-field and fluorescence images of single cells various hours after stimulation using an imaging flow cytometer. Our results indicate distinct temporal dynamics of CD3 and CD28. Notably, CD3 and CD28 relocated on the T-cell surface immediately after stimulation, with CD3 receptors dispersing after 3.5 h, whereas CD28 remained clustered for 7.5 h. These receptor morphological changes precede cytokine production, suggesting their potential as early indicators of T-cell activation.