Characterization of nanoparticle mediated laser transfection by femtosecond laser pulses for applications in molecular medicine

Background: In molecular medicine, the manipulation of cells is prerequisite to evaluate genes as therapeutic targets or to transfect cells to develop cell therapeutic strategies. To achieve these purposes it is essential that given transfection techniques are capable of handling high cell numbers in reasonable time spans. To fulfill this demand, an alternative nanoparticle mediated laser transfection method is presented herein. The fs-laser excitation of cell-adhered gold nanoparticles evokes localized membrane permeabilization and enables an inflow of extracellular molecules into cells. Results: The parameters for an efficient and gentle cell manipulation are evaluated in detail. Efficiencies of 90% with a cell viability of 93% were achieved for siRNA transfection. The proof for a molecular medical approach is demonstrated by highly efficient knock down of the oncogene HMGA2 in a rapidly proliferating prostate carcinoma in vitro model using siRNA. Additionally, investigations concerning the initial perforation mechanism are conducted. Next to theoretical simulations, the laser induced effects are experimentally investigated by spectrometric and microscopic analysis. The results indicate that near field effects are the initial mechanism of membrane permeabilization. Conclusion: This methodical approach combined with an automated setup, allows a high throughput targeting of several 100,000 cells within seconds, providing an excellent tool for in vitro applications in molecular medicine. NIR fs lasers are characterized by specific advantages when compared to lasers employing longer (ps/ns) pulses in the visible regime. The NIR fs pulses generate low thermal impact while allowing high penetration depths into tissue. Therefore fs lasers could be used for prospective in vivo applications.

Interaction of ions, electrons, and laser pulses with surfaces

133 p.

Spectral self-action of THz emission from ionizing two-color laser pulses in gases

The spectrum of terahertz (THz) emission in gases via ionizing two-color femtosecond pulses is analyzed by means of a semi-analytic model and numerical simulations in 1D, 2D and 3D geometries taking into account propagation effects of both pump and THz fields. We show that produced THz signals interact with free electron trajectories and thus significantly influence further THz generation upon propagation, i.e., make the process inherently nonlocal. This self-action contributes to the observed strong spectral broadening of the generated THz field. We show that diffraction of the generated THz radiation is the limiting factor for the co-propagating low frequency amplitudes and thus for the self-action mechanism in 2D and 3D geometries.

Do in vivo terahertz imaging systems comply with safety guidelines?

Techniques for the coherent generation and detection of electromagnetic radiation in the far infrared, or terahertz, region of the electromagnetic spectrum have recently developed rapidly and may soon be applied for in vivo medical imaging. Both continuous wave and pulsed imaging systems are under development, with terahertz pulsed imaging being the more common method. Typically a pump and probe technique is used, with picosecond pulses of terahertz radiation generated from femtosecond infrared laser pulses, using an antenna or nonlinear crystal. After interaction with the subject either by transmission or reflection, coherent detection is achieved when the terahertz beam is combined with the probe laser beam. Raster scanning of the subject leads to an image data set comprising a time series representing the pulse at each pixel. A set of parametric images may be calculated, mapping the values of various parameters calculated from the shape of the pulses. A safety analysis has been performed, based on current guidelines for skin exposure to radiation of wavelengths 2.6 µm–20 mm (15 GHz–115 THz), to determine the maximum permissible exposure (MPE) for such a terahertz imaging system. The international guidelines for this range of wavelengths are drawn from two U.S. standards documents. The method for this analysis was taken from the American National Standard for the Safe Use of Lasers (ANSI Z136.1), and to ensure a conservative analysis, parameters were drawn from both this standard and from the IEEE Standard for Safety Levels with Respect to Human Exposure to Radio Frequency Electromagnetic Fields (C95.1). The calculated maximum permissible average beam power was 3 mW, indicating that typical terahertz imaging systems are safe according to the current guidelines. Further developments may however result in systems that will exceed the calculated limit. Furthermore, the published MPEs for pulsed exposures are based on measurements at shorter wavelengths and with pulses of longer duration than those used in terahertz pulsed imaging systems, so the results should be treated with caution.

Role of the range of the dipole function in the classical dynamics of molecular dissociation

The dissociation dynamics of heteronuclear diatomic molecules induced by infrared laser pulses is investigated within the framework of the classical driven Morse oscillator. The interaction between the molecule and the laser field described in the dipole formulation is given by the product of a time-dependent external field with a position-dependent permanent dipole function. The effects of changing the spatial range of the dipole function in the classical dissociation dynamics of large ensembles of trajectories are studied. Numerical calculations have been performed for distinct amplitudes and carrier frequencies of the external pulses and also for ensembles with different initial energies. It is found that there exist a set of values of the dipole range for which the dissociation probability can be completely suppressed. The dependence of the dissociation on the dipole range is explained through the examination of the Fourier series coefficients of the dipole function in the angle variable of the free system. In particular, the suppression of dissociation corresponds to dipole ranges for which the Fourier coefficients associated with nonlinear resonances are null and the chaotic region in the phase space is reduced to thin layers. In this context, it is shown that the suppression of dissociation of heteronuclear molecules for certain frequencies of the external field is a consequence of the finite range of the corresponding permanent dipole. © 2013 American Physical Society.

Quantum-classical correspondence and the role of the dipole function in molecular dissociation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Collimated Field Emission Beams from Metal Double-Gate Nanotip Arrays Optically Excited via Surface Plasmon Resonance

The generation of collimated electron beams from metal double-gate nanotip arrays excited by near infrared laser pulses is studied. Using electromagnetic and particle tracking simulations, we showed that electron pulses with small rms transverse velocities are efficiently produced from nanotip arrays by laser-induced field emission with the laser wavelength tuned to surface plasmon polariton resonance of the stacked double-gate structure. The result indicates the possibility of realizing a metal nanotip array cathode that outperforms state-of-the-art photocathodes.

Blue upconversion luminescence generation in Ce3+: Gd2SiO5 crystals by infrared femtosecond laser irradiation

Blue frequency-upconversion fluorescence emission has been observed in Ce3+-doped Gd2SiO5 single crystals, pumped with 120-fs 800 nm IR laser pulses. The observed fluorescence emission peaks at about 440nm is due to 5d -> 4f transition of Ce3+ ions. The intensity dependence of the blue fluorescence emission on the IR excitation laser power obeys the cubic law, demonstrating three-photon absorption process. Analysis suggested that three-photon simultaneous absorption induced population inversion should be the predominant frequency upconversion mechanism. (c) 2006 Optical Society of America.

Generation of 1.5-12 ns width-tunable 532 nm pulses by adopting laser-induced plasma shutter technique

The pulse-shaping technique has found widespread applications in nonlinear optics and material processing. Experimental research on laser-induced plasma shutter to control the 532 nm pulse width is conducted. The impacts of the total pulse output energy on pulse compression are investigated, and a useful conclusion can be drawn that there exists an optimal value of pulse energy at which the shortest output pulse of 3.23 ns can be obtained without a device for delay-time. Once the device for delay-time is employed to change the optical differences between two laser paths, the pulse width can be further shortened to 1.51 ns. In short, the 1.5-12 ns width-tunable 532 nm laser pulses have been obtained by adopting the laser-induced plasma shutter technique. (C) 2007 Elsevier GmbH. All rights reserved.

Fiber-Optic Ring-Down Spectroscopy Using a Tunable Picosecond Gain-Switched Diode Laser

Visible and near-infrared laser light pulses were coupled into two different types of optical fiber cavities. One cavity consisted of a short strand of fiber waveguide that contained two identical fiber Bragg gratings. Another cavity was made using a loop of optical fiber. In either cavity ∼ 40 ps laser pulses, which were generated using a custom-built gainswitched diode laser, circulated for a large number of round trips. The optical loss of either cavity was determined from the ring-down times. Cavity ring-down spectroscopy was performed on 200 pL volumes of liquid samples that were injected into the cavities using a 100 μm gap in the fiber loop. A detection limit of 20 ppm of methylene blue dye in aqueous solution, corresponding to a minimum absorptivity of εC < 6 cm−1, was realized.

Attosecond phase locking of harmonics emitted from laser-produced plasmas

Laser-driven coherent extreme-ultraviolet (XUV) sources provide pulses lasting a few hundred attoseconds(1,2), enabling real-time access to dynamic changes of the electronic structure of matter(3,4), the fastest processes outside the atomic nucleus. These pulses, however, are typically rather weak. Exploiting the ultrahigh brilliance of accelerator-based XUV sources(5) and the unique time structure of their laser-based counterparts would open intriguing opportunities in ultrafast X-ray and high-field science, extending powerful nonlinear optical and pump-probe techniques towards X-ray frequencies, and paving the way towards unequalled radiation intensities. Relativistic laser-plasma interactions have been identified as a promising approach to achieve this goal(6-13). Recent experiments confirmed that relativistically driven overdense plasmas are able to convert infrared laser light into harmonic XUV radiation with unparalleled efficiency, and demonstrated the scalability of the generation technique towards hard X-rays(14-19). Here we show that the phases of the XUV harmonics emanating from the interaction processes are synchronized, and therefore enable attosecond temporal bunching. Along with the previous findings concerning energy conversion and recent advances in high-power laser technology, our experiment demonstrates the feasibility of confining unprecedented amounts of light energy to within less than one femtosecond.

Photo-ionized neon plasmas induced by radiation pulses of a laser-plasma EUV source and a free electron laser FLASH

In this work, a laser-produced plasma extreme ultraviolet source and a free electron laser were used to create Ne photo-ionized plasmas. In both cases, a radiation beam was focused onto a gas stream injected into a vacuum chamber synchronously with the radiation pulse. Extreme ultraviolet radiation from the plasma spanned a wide spectral range with pronounced maximum centered at lambda = 11 +/- 1 nm while the free electron laser pulses were emitted at a wavelength of 32 nm. The power density of the focused plasma radiation was approximately 2 x 10(7) W/cm(2) and was seven orders of magnitude lower compared with the focused free electron laser beam. Radiation fluences in both experimental conditions were comparable. Despite quite different spectral characteristics and extremely different power densities, emission spectra of both photo-ionized plasmas consist of the same spectral lines within a wavelength range of 20 to 50 nm, however, with different relative intensities of the corresponding lines. The dominating spectral lines originated from singly charged ions (Ne II); however, Ne III lines were also detected. Additionally, computer simulations of the emission spectra, obtained for photo-ionized plasmas, driven by the plasma extreme ultraviolet source, were performed. The corresponding measured and calculated spectra are presented. An electron temperature and ionic composition were estimated. Differences between the experimental spectra, obtained for both irradiation conditions, were analyzed. The differences were attributed mainly to different energies of driving photons.

Production of defects in ZBLAN, ZBLAN : Tm3+ and ZBLAN : Cr3+ glasses by ultra-short pulses laser interaction

In this work we have studied pure and thulium- and chromium-doped ZBLAN glasses irradiated by ultra-short laser pulses. A Ti:sapphire CPA system was used, producing a 500 Hz train of pulses, centered at 830 nm, with 375 mu J of energy and 50 fs of duration (FWHM). The beam was focused by a 20 Him lens, producing a converging beam with a waist of 12 pin. The absorption spectra before and after laser irradiation were obtained showing production of color centers in pure, thulium-doped and chromium-doped ZBLAN glasses. A damage threshold of 9.56 TW/cm(2) was determined for ZBLAN. (C) 2007 Elsevier Ltd. All rights reserved.