Figure 4.

SPT of membrane and cytoplasmic proteins using the DHPSF in live human T cells. (a) Experimental procedure for SPT at the apical surface using the DHPSF. Jurkat T cells were labeled with HaloTag-TMR and dropped onto passivating-coated coverlips. A 60x water immersion objective was focused at the apical surface to image the membrane protein TCR or the cytoplasmic protein Zap70. TCR was also imaged in fixed cells. (b) Representative side-on (top) and top-down (bottom) views of trajectories of unfixed TCR (left) and Zap70 (right) proteins at the apical surface. The difference in speed between the two proteins can be clearly seen in the length of trajectories. (c) Mean diffusion coefficient for Zap70 and TCR live and fixed determined by MSD analysis (horizontal bars) with cell-to-cell variation (circles) and total number of trajectories. The size of the circles is proportional to the number of tracks obtained from the cell. Small circles represent cells with fewer trajectories while large circles represent cells with many trajectories. The number of tracks ranges from ∼30-140 per cell for Zap70 and ∼5-140 for TCR.