The T cell receptor (TCR) is the key membrane protein that participates in the activation of T cells in response to the recognition of antigens presented by the major histocompatibility complex (pMHC) on the membrane of antigen presenting cells. This binding process is likely to depend on the dynamics of the participating membrane proteins, making the analysis of their diffusion an important task for studying TCRpMHC interaction. The previously described low mobility of the TCR (D 0.05 m/s) might impair the efficiency of TCR-pMHC engagement, encouraging the task to verify if the slow mobility is indeed inherent to T cells or if it results from external cell treatments required for microscopy approaches. In this work, single molecule tracking experiments were performed to investigate to which degree the diffusion behavior of the TCR is affected by different experimental conditions. Tracking experiments were done using fluorescently labelled anti-TCR single chain fragments (scFV) derived from the monoclonal antibody H57. Three different strategies for presenting adhesive surfaces to T cells were compared: (i) the homo-polymer Poly-D-Lysine, (ii) the glycoprotein Fibronectin and (iii) a supported lipid bilayer presenting intercellular adhesion molecule 1 (ICAM-1). Furthermore, Highly Inclined and Laminated Optical Sheet (HILO) microscopy was utilized to compare the diffusion on the apical membrane to the results derived from Total Internal Reflection (TIR) imaging on the basal T cell membrane. To further exclude fluorophore influences on the diffusion measurements, scFV were conjugated to varying organic dyes and the diffusion results were compared. Similar diffusion characteristics of the TCR were found on all three coated surfaces, with D ranging from 0.03 0.06 m/s. Also, different fluorophores attached to the scFV or the usage of full antibodies instead of the single chain fragment yielded similar results. Furthermore, TCR mobility at the top membrane measured via HiLO microscopy was similar to the mobility at the bottom membrane measured via TIRF microscopy. From this data we conclude that there is no direct influence of the adhesive surfaces on the mobility of the TCR. In the second part of this work the interaction kinetics of the - and TCR subunits of the TCR/CD3 complex were measured using different imaging modalities. Recent measurements have indicated discrepancies in the dynamic behavior of these components, reporting a faster diffusion for . To address these findings, diffusion properties were obtained from single particle tracking experiments, and further investigated by combining in vivo micropatterning with Fluorescence Recovery after Photobleaching (FRAP). The diffusion analysis of the tracking measurements hints to a more mobile -chain, highlighting the possibility of TCR/CD3-independent within the T cell membrane. Evaluation of the signal recovery curve from the FRAP experiments indicates a stable interaction between TCR/CD3 subunits. While the FRAP results do not represent a more mobile chain, they also do not exclude the existence of free membrane-bound .