Optoinjection efficiency differs depending upon the osmolarity of the surrounding medium.

<p>From looking at A, as we increase the osmolarity, the efficiency of optoinjection (black squares) increases from zero up to 50% as the surrounding medium changes from hypertonic to hypotonic. Conversely, the optoejection efficiency (red circles) falls from around 10% to 0% above 320±2 mOsm/L. As the molarity of the solution increases, the likelihood of cell death (represented by blue triangles) is increased. At 320±2 mOsm/L the optoinjection and ejection efficiency are approximately equal. Each data point was performed in triplicate with 20 cells; error bars represent S.E.M. B and C show brightfield and fluorescence overlays of a cell prior to (B) and 2 minutes after (C) laser irradiation in standard culture medium in the presence of PI. Photoporation induces a flux of cytosol from the cell into the extracellular medium. The cytosol ejected from the cell becomes stained with PI and a bright fluorescence is seen exterior to the cell. Large changes in cell morphology occur and cell death is induced. D shows the osmotic effect on plasmolysis of BY-2 cells. Both the void space (blue) and fraction of plasmolyzed cells (red) increases with the osmolarity of the surrounding medium but the red line shows a much steeper incline around the point of incipient plasmolysis (50%). Error bars denote the S.E.M. for n = 3 experiments with 20 cells counted in each. E-G show example cells in standard culure medium (171±2 mOsm/L), very weakly hypotonic (320±2 mOsm/L) and strongly hypotonic (699±4 mOsm/L) solutions respectively, with the resulting plasmolysis occurring slightly in F but seen very strongly in G as the membrane pulls away from the cell wall in the highly osmotic solution. Scale bars denote 10 µm.