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.</p

Optoinjection efficiency (O) and viability (V) of the BY-2 plant cells at different laser powers.

<p>After irradiation by (A) three shots or (B) a single shot with the Gaussian beam or (C) a single shot with the Bessel beam. For both beam geometries the optoinjection efficiency (represented by open squares) increases with power at the focal plane while viability (solid squares) usually decreases. C shows N (the proportion of cells being both viable and optoinjected) for varying central spot intensities. N increases as the intensity increases. The Bessel beam (black) shows a higher value for N than the Gaussian beam (red) when considering a single shot. When comparing with three axially separated shots of the Gaussian beam (blue), N is comparable to the Bessel beam. Each data point represents the mean for n = 5 with 20 cells per experiment. Error bars represent the standard error of the mean (S.E.M.).</p

Optical injection of PI into a BY-2 cell.

<p>Shown in bright field (A–C) and fluorescence (D–F). A) Before shooting, B) transient bubble created on cell membrane during laser dose, C) no visible laser damage left post-irradiation. D) Pre-irradiation showing faint PI staining of the cell wall. E) Laser induced transient auto-fluorescence at the point of irradiation. F) Permanent increase in cytosolic fluorescence as PI enters the cell. Arrows indicate site of laser irradiation. Scale bar denotes 10 µm.</p

Effect of molecule size on cellular uptake.

<p>From looking at A, it can be seen that as the Stokes radius increases, the amount of dextran taken up by the cell decreases. The protoplasts take up less dextrans than the intact cells for Stokes radii smaller than 5 = 30 cells with error bars representing the S.E.M. B shows representative images (those depicting uptake most comparable to the average uptake) of intact cells and protoplasts before (1) and 3 mins after (2) photoporation in the presence of small and large dextrans. The larger dextrans show less (though still visible) entry into the cell than the smaller dextrans.</p

Uptake of calcein during photoporation in hypertonic medium.

<p>Solid lines in A show the mean increase in intracellular fluorescence relative to background for n = 20 cells with error bars representing 0.5 S.E.M. for clarity. Each curve shows a sharp increase in fluorescence in the first few minutes after photoporation, which plateaus after around 3 minutes. The higher molarity solutions show a quicker increase in calcein uptake and reach a higher level of maximum fluorescence than the lower molarity solutions. Dashed lines denote fitted saturation curves. B and C show a cell (arrowed), in negative contrast for clarity, before and 60 seconds after photoporation in 0.4 Osm/L medium containing calcein. The nucleus, indistinguishable from the rest of the unporated cell in B, becomes filled with calcein along with a cytosolic strand (just visible at arrow tip) but none enters the large vacuole surrounding it.</p

Optical set-up applied for plant cell photoporation.

<p>A provides a series of schematics depicting Gaussian and Bessel beams. The first column shows example microscope images of transverse profiles of focused Gaussian (i) and Bessel (ii) beams. Illustrative axial profiles are seen in iii and iv where the regions of multi-photon absorption are shown in red. The Bessel beam axial extent of multi-photon absorption is much longer than that of the Gaussian. The white dotted lines show the corresponding cross-sections of the orthogonal images. The final column experimentally shows two-photon excitation of fluorescein of each beam adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079235#pone.0079235-Brown1" target="_blank">[30]</a>, scale bars represent 100 µm. Laser setup is shown in B. Output from the laser was directed into either of two arms using a removable mirror, denoted by dotted lines. A Bessel beam was generated using an axicon and a Gaussian beam spot was created using a system of telescopes (L1 and L2). The beam paths were directed into the back of a commercial inverted microscope where all imaging was performed.</p

Parameters determined from non-linear fits of calcein uptake.

<p>As the osmolarity increases, the asymptotic maximum fluorescence value, which the saturation curve tends towards, increases. The time taken to reach 50% of the maximum fluorescence decreases as the molarity of the solution increases. The increased pressure difference caused by higher molarity solutions induces more and quicker uptake of medium to balance it. Brackets denote the error in the final digit; uncertainties were calculated from the R-squared value of the fitted curves.</p