Figure 1

(a) Schematic of optical setup. The femtosecond near-infrared laser is coupled into the laser port of an inverted fluorescence microscope. The laser beam diameter is expanded from 1.7 to $10\mathrm{mm}$ by two lenses with focal length $f=38\mathrm{mm}$ and $f=200\mathrm{mm}$, respectively. The laser power is controlled by a variable attenuator and measured by a laser power meter. The exposure time of the cell samples to the laser beam is controlled by a programmable shutter. The experiment is observed by a video camera. Images are stored on a personal computer. (b) Schematic of the laser beam path inside the inverted fluorescence microscope. A dichroic mirror (laser filter) in the upper filter wheel directs the laser beam through the objective onto the cell sample. The filter cubes for the fluorescence microscopy are in the lower filter wheel.Reuse & Permissions

Figure 2

Illustration of the principle of dye uptake experiments. Cells are cultured in media containing membrane impermeable fluorescent dyes. The laser focal spot is directed onto the cell membrane. Cell membrane permeabilization by the laser beam is probed by observing dye uptake by the cell.Reuse & Permissions

Figure 3

(a) Left: The picture shows a BAEC cell before laser exposure in the presence of Lucifer Yellow. The laser focal spot is marked by the white circle. (a) Right: The picture shows BAEC cells after laser exposure in the presence of Lucifer Yellow. After laser exposure fluorescence is observed inside the BAEC cell. (b) The same experiment has been performed with PI as fluorescent probe. Again the location of the laser focal spot is indicated by a white circle. Again, after laser exposure fluorescence is observed inside the cell.Reuse & Permissions

Figure 4

(a) Target BAEC cell after optoinjection. The location of the laser focal spot is indicated by a black circle. The region selected for time-lapse analysis is indicated by a white circle. The illumination sites and the observation sites are always in constant distance. (b) Time-lapse analysis of dye (Lucifer Yellow) uptake process. The fluorescence intensity emitted by the selected region inside the cell starts to increase when the laser shutter is opened at time $t=18\mathrm{s}$ and the cell is exposed to the beam. During laser exposure the fluorescence intensity from the selected region increases linearly with time. After the laser shutter is closed at time $t=38\mathrm{s}$ a further sublinear increase of the fluorescence intensity is observed until the fluorescence intensity measured in the selected region reaches a constant value at time $t=240\mathrm{s}$. The laser intensity was $6.4\times {10}^{12}\mathrm{W}∕{\mathrm{cm}}^2$. The linear increase in fluorescence intensity in the selected region inside the cell during laser exposure is a measure of the number of dye molecules diffusing into the cell through the transient pore created by the fs nir laser beam. The linear slope of the time-lapse record during laser exposure is the rate of the diffusive flow $\Phi \sim dN∕dt$ of dye molecules into the cell.Reuse & Permissions

Figure 5

The graph shows the rate of increase of average fluorescent intensity under different laser intensities in optoinjection experiment performed with BAEC and Lucifer Yellow. The rate of increase of fluorescence intensity is the linear slope of the time-lapse records during laser exposure (Fig. 4). The linear slope of time-lapse records during laser exposure are rates of diffusive flow $\Phi \sim dN∕dt$ of dye molecules into the cell. We have performed optoinjection experiments on living BAEC at ten different laser intensities $I_i$. Furthermore, we have performed optoinjection experiments on 20 individual cells for a given laser intensity $I_i$. The total number of BAEC cells investigated in our study was 200 BAEC cells. For each individual cell, the rate of increase of fluorescence intensity was determined from the corresponding time-lapse record. The data points displayed are the statistical means of the rate of increase of fluorescence intensity observed for 20 living cells investigated at that laser intensity $I_i$. The vertical error bars are the standard error of the mean rate of increase of fluorescence intensity. The statistical analysis was performed using the descriptive statistics software package in the data analysis software ORIGIN 7.0. We do not observe dye uptake for laser intensity levels less than $4.0\times {10}^{12}\mathrm{W}∕{\mathrm{cm}}^2$. For laser intensities between $4.0\times {10}^{12}\mathrm{W}∕{\mathrm{cm}}^2$ and $1.0\times {10}^{13}\mathrm{W}∕{\mathrm{cm}}^2$ we observe a controlled increase of the rate of dye uptake with increasing laser intensity. The vertical error bars indicate the cell-to-cell variations in rate of dye uptake for a given laser intensity. For laser intensities between $1.0\times {10}^{13}\mathrm{W}∕{\mathrm{cm}}^2$ and $3.5\times {10}^{13}\mathrm{W}∕{\mathrm{cm}}^2$ we observe larger cell-to-cell variations than for laser intensities below $1.0\times {10}^{13}\mathrm{W}∕{\mathrm{cm}}^2$. For laser power levels larger than $3.5\times {10}^{13}\mathrm{W}∕{\mathrm{cm}}^2$ we observe disintegration of the cell. The solid line is a fit of $\Phi =A\ln (I_i∕B)$ to the measured data with $A=6.02\pm 1.13$ and $B=(2.62\pm 0.87)\times {10}^{12}\mathrm{W}∕{\mathrm{cm}}^2$. This logarithmic dependence of $\Phi$ on $I_i$ is derived assuming laser-induced plasma formation in the laser focal spot and considering the Gaussian intensity distribution in the laser focal spot.Reuse & Permissions

Figure 6

Gaussian intensity distribution $I\left(r\right)$ in the laser focal spot for various laser intensities under fixed focusing conditions. The horizontal solid line indicates a line of constant intensity $I_C$.Reuse & Permissions