In this paper, we present the results of in vitro gene transfer by plasmonic enhanced optoporation of human melanoma
cells. The fs-laser based optoporation is a gentle and efficient method for transfection. An optimum perforation rate with
efficient dye or DNA uptake and high viability of the cells (~90%) was found for different types of nanostructures,
spherical and rod shaped. The technique offers a very high selectivity and the low damage induced to the cell leads to a
high transfection efficiency. The cell selectivity of this technique on the one hand is realized by using bioconjugated
nanostructures, that couple selectively to a special cell type, and on the other hand, the spatial selectivity is due to the
fact that only irradiated cells are perforated. In many biological applications a virus free and efficient transfection
method is needed, especially in terms of its use in vivo. In cancer cells, the aggressiveness of the cells is shown in the
migration and invasion velocity. The laser based and nanostructure enhanced transfection of cells offers the possibility to
directly compare the treated and untreated cells. The treatment for migration and invasion assays can be performed by
laser-scraping and laser transfection, resulting in a fully non-contact and therefore sterile method where the shape and
the size of the scrape is well defined and reproducible. The laser based scrape test therefore offers less uncertainty due to
scrape variations, high transfection efficiency, as well as direct comparison of treated and control cells in the same dish.
The gold nanoparticle (AuNP) mediated ultrashort laser cell membrane perforation has been proven as an efficient
delivery method to bring membrane impermeable molecules into the cytoplasm. Nevertheless, the underlying
mechanisms have not been fully determined yet. Different effects may occur when irradiating a AuNP with ultrashort
laser pulses and finally enable the molecule to transfer. Depending on the parameters (pulse length, laser fluence and
wavelength, particle size and shape, etc.) light absorption or an enhanced near field scattering can lead to perforation of
the cell membrane when the particle is in close vicinity. Here we present our experimental results to clarify the
perforation initiating mechanisms. The generation of cavitation and gas bubbles due to the laser induced effects were
observed via time resolved imaging. Additionally, pump-probe experiments for bubble detection was performed.
Furthermore, in our patch clamp studies a depolarization of the membrane potential and the current through the
membrane of AuNP loaded cell during laser treatment was detected. This indicates an exchange of extra- and intra
cellular ions trough the perforated cell membrane for some milliseconds. Additionally investigations by ESEM imaging
were applied to study the interaction of cells and AuNP after co incubation. The images show an attachment of AuNP at
the cell membrane after several hours of incubation. Moreover, images of irradiated and AuNP loaded cells were taken to
visualize the laser induced effects.
Femtosecond (fs) laser-based cell surgery is typically done in two different regimes, at kHz or MHz repetition rate. Formation of reactive oxygen species (ROS) is an often predicted effect due to illumination with short laser pulses in biological tissue. We present our study on ROS formation in single cells in response to irradiation with fs laser pulses depending on the repetition rate while focusing into the cell nucleus. We observed a significant increase of ROS concentration directly after manipulation followed by a decrease in both regimes at kHz and MHz repetition rate. In addition, effects of consecutive exposures at MHz and kHz repetition rate and vice versa on ROS production were studied. Irradiation with a MHz pulse train followed by a kHz pulse train resulted in a significantly higher increase of ROS concentration than in the reversed case and often caused cell death. In the presence of the antioxidant ascorbic acid, accumulation of ROS and cell death were strongly reduced. Therefore, addition of antioxidants during fs laser-based cell surgery experiments could be advantageous in terms of suppressing photochemical damage to the cell.
We present our results for nanoparticle mediated laser
poration as an alternative transfection technique. As a fundamental part for the perforation of the cell membrane the interactions of gold nanoparticles and living cells were studied.
Investigation on the interaction of small particles, e.g. gold nanoparticles with light is a current field of high interest. As
light can be absorbed, enhanced or scattered by the nanoparticles a wide variety of possible applications become
possible. If the electrons of such a nanoparticles oscillate with the incident light, plasmon resonances occur. Provided
that these particles are brought very close to a cell, the cell membrane gets perforated due to the laser induced effect. We
investigate nanoparticle mediated laser perforation as an alternative technique for cell transfection. By using weakly
focussed femtosecond laser pulses, 150 nm gold particles were stimulated to perforate the cell membrane. Through the
perforated area of the membrane macromolecules e.g. DNA are able to enter the cell. By this technique GFSHR-17 rat
cells were successfully transfected with GFP vector and the dependence on laser parameters and concentration were
studied. Even after 48 hours after manipulation the transfected cells show no indications of apoptosis or necrosis. This
technique allows the transfection of cells by opto-perforation without the need of tight focusing conditions and single
cell targeting- opening the way for a wide field of applications.
The manipulation of cells by femtosecond (fs) laser pulses became a very important tool in cell biology. In terms
of learning more about the function of the cell compartments and the cell dynamics, single cell organelles are
manipulated by laser pulses. Meanwhile the cell reaction is observed by different microscopy methods. The
parameters of the laser irradiation have to be chosen carefully to minimize unwanted side effects during the
treatment and to prevent cell damage or cell death. In many applications, it is not known what happens due to
the laser irradiation on the molecular level. The formation of reactive oxygen species (ROS) is an often predicted
effect due to photo disruption in biologic tissue. In this paper, we present our study of the ROS formation during
the irradiation of fs laser pulses for disruption of single cell organelles. The quantity of ROS formation depends
strongly on the pulse energy of the laser. Therefore the creation of ROS was additionally studied while scanning
the laser at low energy for multiphoton microscopy.
Fluorescence imaging of cells and cell organelles requires labeling by fluorophores. The labeling of living cells is often done by transfection of
fluorescent proteins. Viral vectors are transferring the DNA into the cell. To avoid the use of viruses, it is possible to perforate the cell
membrane for example by electro-shocks, the so called electroporation, so that the fluorescent proteins can diffuse into the cell. This method
causes cell death in up to 50% of the treated cells because the damage of the outer membrane is too large.
A less lethal perforation of the cell membrane with high efficiency can be realized by femtosecond (fs) laser pulses. Transient pores are created by focusing the laser beam for some milliseconds on the membrane. Through this pore, the proteins can enter into the cell. This was demonstrated in a proof of principle experiment for a few cells, but it is essential to develop an opto-perforation system for
large numbers of cells in order to obtain statistically significant samples for biological experiments. The relationship between pulse energy,
irradiation time, repetition rate and efficacy of the transfer of a chromophor into the cells as well as the viability of the cells was analysed. The cell viability was observed up to 90 minutes after manipulation.
Multiphoton microscopy is a very promising method for 3D imaging
of living cells. The fluorochromes are solely excited at the laser
focus by multiphoton absorption using near-infrared femtosecond
laser pulses. The arising fluorescence serves for a pixel-to-pixel
imaging with a resolution in the submicron range. At higher laser
powers, the multiphoton absorption creates a micro plasma which
induces an outwardly propagating shock wave. The rapidly expanding
cavitation bubble causes disruption of the material, with hardly
any interaction with the surrounding tissue as the optical breakdown proceeds faster than the thermal conduction.
This combination offers the possibility of simultaneous manipulation and analysis of living cells or cell organelles. Manipulation is achieved using laser pulses with an energy of a few nanojoules while imaging is done at less than 1 nJ. The obtained resolution allows the precise cutting of single cell organelles without compromising the cells` viability. Thus, the implementation is excellently suited for cell surgery. We conducted ablation of different subcellular structures, like mitochondria, at different pulse energies within living cells while studying cell viability.
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