Figure 2: Two-photon guided T-cell migration in vitro.

(a) Time-lapse images of a dsRed-expressing CD8⁺ T cell chasing the focus of a 720-nm two-photon excitation laser in media containing 1 μg ml⁻¹ hCCL5**. The uncaging laser focus was manually placed in front of the leading edge. Green dots: previous positions of the laser focus; green cross: current focus at the indicated time point; red dots: centroid positions of the cell. Scale bar, 20 μm. (b) Time-lapse images showing T-cell accumulation over time towards the uncaging light path patterned in a heart shape (red line) in hCCL5**-containing media. Scale bar, 50 μm. Data representative of more than five independent experiments. Time-lapse images showing a stable (c) or unstable (d) contact engaged CD8⁺ T-cell response to hCCL5 attraction delivered by a focus of 720-nm two-photon excitation continuously. Red dots: previous positions of the laser focus; red cross: current focus at the indicated time point. Scale bar, 5 μm. Data are representative of more than five independent experiments.