J. Baumgart

Quantified femtosecond laser based opto-perforation of living GFSHR-17 and MTH53 a cells.

Opto-perforation is an interesting alternative to conventional techniques for gene transfer into living cells. The cell membrane is perforated by femtosecond (fs) laser pulses, in order to induce an uptake of macromolecules e.g. DNA. In this study, we successfully transfected a canine cell line (MTH53a) with GFP vector or a vector coding for a GFP-HMGB1 fusion protein. The transfected cells were observed 48 hours after treatment and they were not showing any signs of apoptosis or necrosis. Based on simultaneously measured membrane potential changes during the perforation, we were able to calculate and experimentally verify that the relative volume exchanged is 0.4 times the total cell volume. Thus, for first time a quantitative predication of the amount of uptaken molecules and therefore a quantification of the transfection is possible. Additionally, this method offers new high efficient possibilities for critical transfection approaches involving special cell types, e.g. primary and stem cells.

TNF-α induced secretion of HMGB1 from non-immune canine mammary epithelial cells (MTH53A)

BACKGROUND Mammary neoplasias are one of the most frequent and spontaneously occurring malignancies in dogs and humans. Due to the similar anatomy of the mammary gland in both species, the dog has become an important animal model for this cancer entity. In human breast carcinomas, the overexpression of a protein named high-mobility group box 1 (HMGB1) was reported. Cells of the immune system were described to release HMGB1 actively exerting cytokine function. Thereby it is involved in the immune system activation, tissue repair, and cell migration. Passive release of HMGB1 by necrotic cells at sites of tissue damage or in necrotic hypoxic regions of tumors induces cellular responses e.g. release of proinflammatory cytokines leading to elevated inflammatory response and neo-vascularization of necrotic tumor areas. Herein we investigated if a time-dependent stimulation with the separately applied proinflammatory cytokines TNF-α and IFN-γ can cause secretion of HMGB1 in a non-immune related HMGB1-non-secreting epithelial canine mammary cell line (MTH53A) derived from non-neoplastic tissue. METHODS The canine cell line was transfected with recombinant HMGB1 bicistronic expression vectors and stimulated after transfection with the respective cytokine independently for 6, 24 and 48 h. HMGB1 protein detection was performed by Western blot analysis and quantified a by enzyme-linked immunosorbent assay. Live cell laser scanning multiphoton microscopy of MTH53A cells expressing a HMGB1-GFP fusion protein was performed in order to examine, if secretion of HMGB1 under cytokine stimulating conditions is also visible by fluorescence imaging. RESULTS The observed HMGB1 release kinetics showed a clearly time-dependent manner with a peak release 24h after TNF-α stimulation, while stimulation with IFN-γ had only small effects on the HMGB1 release. Multiphoton HMGB1 live cell microscopy showed diffuse cell membrane structure changes 29 h after cytokine-stimulation but no clear secretion of HMGB1-GFP after TNF-α stimulation was visible. CONCLUSION Our results demonstrate that non-immune HMGB1-non-secreting cells of epithelial origin derived from mammary non-neoplastic tissue can be induced to release HMGB1 by single cytokine application. This indicates that tumor and surrounding tissue can be stimulated by tumor present inflammatory and necrotic cytokines to release HMGB1 acting as neo-vascularizing factor thus promoting tumor growth.

Repetition rate dependency of reactive oxygen species formation during femtosecond laser-based cell surgery.

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.

Plasmonic perforation of living cells using ultrashort laser pulses and gold nanoparticles

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.

Fs-laser scissors for photobleaching, ablation in fixed samples and living cells, and studies of cell mechanics.

The use of ultrashort laser pulses for microscopy has steadily increased over the past years. In this so-called multiphoton microscopy, laser pulses with pulse duration around 100 femtoseconds (fs) are used to excite fluorescence within the samples. Due to the high peak powers of fs lasers, the absorption mechanism of the laser light is based on nonlinear absorption. Therefore, the fluorescence signal is highly localized within the bulk of biological materials, similar to a confocal microscope. However, this nonlinear absorption mechanism can not only be used for imaging but for selective alteration of the material at the laser focus: The absorption can on one hand lead to the excitation of fluorescent molecules of fluorescently tagged cells by the simultaneous absorption of two or three photons or on the other hand, in case of higher order processes, to the creation of free-electron plasmas and, consequently, plasma-mediated ablation. Typical imaging powers are in the range of tens of milliwatts using 100-fs pulses at a repetition rate of 80-90 MHz, while pulse energies needed for ablation powers are as low as a few nanojoules when using high numerical aperture microscope objectives for focusing the laser radiation into the sample. Since the first demonstration of this technique, numerous applications of fs lasers have emerged within the field of cellular biology and microscopy. As the typical wavelengths of ultrashort laser systems lie in the near infrared between 800 and 1000 nm, high penetration depth can be achieved and can provide the possibility of imaging and manipulating the biological samples with one single laser system.

Mechanisms of gold nanoparticle mediated ultrashort laser cell membrane perforation

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.

Fs-laser-induced Ca 2+ concentration change during membrane perforation for cell transfection

Fs-laser based opto-perforation is a gentle method for gene transfer into sensitive cells such as stem cells or primary cells. The high selectivity and the low damage to the cell lead to a high efficiency of transfection. However, there are side effects which induce stress to the cell due to the exchange of intra- and extracellular media as well as the disintegration of the structure of biomolecules resulting from the laser exposure. Moreover, the mechanisms of the optical transfection are still unclear. In this paper, we present our study on calcium (Ca(2+)) homeostasis during cell surgery, especially during laser induced membrane perforation. We show that the manipulation of cells can induce an increase in the cytosolic Ca(2+) concentration. This increase was not observed if the manipulation of the cells was performed in absence of the extracellular calcium indicating the importance of the Ca(2+) uptake. We found, that the uptake of extracellular Ca(2+) strongly depends on the repetition rate and the irradiation time of the laser pulses. The exposure for several seconds to kHz pulses even induces Ca(2+) induced Ca(2+) release. Dependent on the location of perforation, probably in the vicinity of an intracellular Ca(2+) stock, an instantaneous intracellular Ca(2+) release can be induced. Since Ca(2+) could be involved in negative side effect by cell surgery, we propose an application of the optoperforation technique in nominal Ca(2+)-free external solution.

Live cell opto-injection by femtosecond laser pulses

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.

Nanoparticle mediated laser cell perforation

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 of reactive oxygen species formation in living cells during femtosecond laser based cell surgery

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.