Figure 2.

Effect of uncoupling trap from imaging on the 3D image reconstruction of a trapped object. In (A) and (B), on the left panels, a single latex bead (arrow) from the ones settled down on the cover glass is trapped, moved by the z-top plate and imaged in brightfield (scale bar, 10 μm); on the right panels, a CD45 membrane-stained T cell hybridoma (CD4 3A9 T cell) was trapped and observed by confocal imaging (stack of 20 images at 1 μm intervals). Scale bar, 15 μm. (A) The sample was moved by the z-top plate without synchronization (Fig. 1B, left panel). The trapped bead stays in the focal plane, whereas beads on the cover glass are defocused. As a consequence, a trapped object is constantly viewed through the identical focal plane all along the stack as illustrated on the right panel with a 3D-image of a cell. (B) Same experiments but with the trapped object maintained in its reference position by synchronization (Fig. 1B, right panel). The trapped bead appears defocused as the ones on the cover glass. On the right, 3D image reconstruction of a trapped cell becomes possible from the stack of images recorded at different altitudes. See also Movie S1 and Movie S2, on beads and cells, respectively.