Cell phase volume change evaluated by holographic microscopy.

<p>a) Phase images show phase volume and area change after 40 ms irradiation time for two radiant exposures. See corresponding supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.s005" target="_blank">S1 Video</a>. Scale bars are 20 <i>μ</i>m. b) Shown here are box plots of the fit parameters for the relative phase volume change of cells over a time of 60 s. See supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.s007" target="_blank">S3 Video</a>. Whiskers for box plots depict one standard deviation. The upper box plot indicates that the total loss of phase volume increases with radiant exposure. The middle box plot presents the linear slope of the fit, which is an indicator of the slow process of cell phase volume change. It can be either positive or negative, corresponding to linear phase volume increase or decrease. In the bottom box plot, the exponential decay parameter is depicted. The percentage of cells, which show a combination of linear and exponential decay is indicated in the upper histogram panel. The graph below this shows the decay constants for those cells which exhibited an exponential volume decay after irradiation. The decay parameter is smallest on average for highest radiant exposure, suggesting fastest loss of volume. Data of at least 22 irradiated cells is shown.</p

Potential causes of the calcium signaling during gold nanoparticle mediated laser manipulation.

<p>Two different pathways in the presence or absence of a calcium chelating agent like EGTA are depicted. The intercellular calcium wave occurs in the presence of EGTA because the cascade is possibly mediated by the IP₃ pathway or paracrine ATP signaling. ATP might also be released from the cell after perforation.</p

Cell area evaluated by holographic microscopy after laser manipulation.

<p>Cell area a) 30 s and b) 60 s post irradiation is normalized to the cell area before laser manipulation. Box plots indicate the distribution of the area for the irradiation times and radiant exposures under examination. The unirradiated control is based on the same dataset in both diagrams. The highest radiant exposure of 41 mJ/cm² led to a diversified area distribution, while 27 mJ/cm² mainly caused an area decrease. In the case of 15 mJ/cm² the area distribution was similar to irradiated cells. Data of at least 22 irradiated cells is shown.</p

Schematic of the experimental setup.

<p>An epifluorescence microscope is modified to capture digital holography and fluorescence image data. Coherent illumination necessary for digital holography is realized by weakly focusing a cw Helium Neon Laser on the sample. The pulsed manipulation laser is coupled into the setup parallel to the HeNe-Laser using a dichroic mirror (DM) and focused onto the sample. For fluorescence excitation a mercury vapor lamp is employed. At the side port of the microscope the manipulation laser is attenuated using a notch filter (F1). A beam splitter directs 90% of the light to the fluorescence unit where it passes through a filter blocking the coherent illumination (F2) and an emission filter (F3). The remaining light travels to the digital holography module which is set up in off-axis Michelson configuration.</p

Normalized fluorescence of calcium indicator Fluo-4 with and without addition of calcium chelator EGTA.

<p>a) Fluorescence images at different points in time referring to supplemental video <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.s006" target="_blank">S2 Video</a>. Scale bar 50 <i>μ</i>m. The color map calibration is different for both columns. b) Time-dependence of Fluo-4 intensity relative to its initial value after laser manipulation at zero seconds. 5 mM EGTA was used as calcium chelator in RPMI 1640. Radiant exposure was fixed to 27 mJ/cm². The peak increase with 5 mM EGTA compared to normal conditions is lowered. Signal decrease below base level is most likely explained by photobleaching. The mean and standard error of three experiments with five irradiated and analyzed cells are shown.</p

Normalized fluorescence of calcium indicator Fluo-4 after laser perforation.

<p>Fluorescence intensity is normalized to the value before laser manipulation. The point in time of laser manipulation was set to zero seconds. The increase of Fluo-4 fluorescence intensity after laser manipulation corresponds to an increase in intracellular calcium, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124052#pone.0124052.g007" target="_blank">Fig 7</a>, as well as inflow of extracellular calcium. The process is highly dependent on radiant exposure and irradiation time. Signal decrease below base level is most likely explained by photobleaching. The mean and standard error of three experiments with five irradiated and analyzed cells are shown.</p

Cell viability (a) and perforation efficiency (b) for different radiant exposures and irradiation times.

<p>Viability was evaluated with a calcein/propidium iodide assay one hour after perforation. The perforation efficiency was analyzed via uptake of propidium iodide one minute after laser perforation and is the product of perforated and viable cells. It decreases with higher radiant exposures. Highest perforation efficiency is achieved with 10 ms exposure time and 27 mJ/cm². The mean and standard deviation of three independent measurements are shown. Each measurement consisted of thirty irradiated cell areas.</p

Time series showing the coherency of F-actin in irradiated ZMTH3-Act cells.

<p>For visualization a mean filter of three pixels was applied. A coherency of 1 means a high degree of orientation of local image features. A coherency of zero indicates no orientation. Laser parameter: 10 ms irradiation time and radiant exposure of 41 mJ/cm². Scale bar 50 <i>μ</i>m.</p

siRNA injection using GNOME laser transfection.

<p>A: Transfection of AlexaFluor488 labeled siRNA into ZMTH3 cells. 88% of the cells stained positive for the siRNA after transfection with the optimized parameters (0.5 µg/cm² AuNP, 20 mJ/cm², 50 mm/s). B: Exemplary images of ZMTH3 cells transfected with AlexaFlour488-siRNA and control cells. Scale bar: 100 µm.</p

Experimental setup.

<p>A: Schematic drawing and B: photograph of the setup.</p