B. Agate

Femtosecond optical transfection of cells:viability and efficiency

Photoporation is a rapidly expanding technique for the introduction of macromolecules into single cells. However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm. Photoporation of 4000 Chinese Hamster ovary cells was performed, representing the largest optical transfection study reported to date. We have investigated a range of laser fluences at the cell membrane and, at 1.2 microJ/cm(2), have found an average transfection efficiency of 50 +/- 10%. Contrary to recent literature, in which 100% efficiency is claimed, our measure of efficiency accounts for all irradiated cells, including those lost as a result of laser treatment, thereby providing a true biological measure of the technique.

Femtosecond cellular transfection using a nondiffracting light beam

The ability to permeate selectively the cell membrane and introduce therapeutic agents is a key goal in cell biology. Optical transfection is a powerful methodology but requires exact focusing due to the required two-photon power density. The authors use a Bessel beam that obviates the need to locate precisely the cell membrane, permitting two-photon excitation along a line leading to cell transfection. Assuming a minimum efficiency of 20%, the Bessel beam offers transfection at axial distances 20 times greater than that of its Gaussian equivalent. Furthermore, the authors demonstrate cell transfection beyond obstacles due to the self-healing nature of the Bessel beam.

Femtosecond optical tweezers for in-situ control of two-photon fluorescence

We perform a comparison of optical tweezing using continuous wave (cw) and femtosecond lasers. Measurement of the relative Q-values in the femtosecond and cw regimes shows that femtosecond optical tweezers are just as effective as cw optical tweezers. We also demonstrate simultaneous optical tweezing and in-situ control of two-photon fluorescence (at 400nm) from dye-doped polymer microspheres. By switching the 800 nm tweezing laser source between femtosecond and cw regimes, we turned the fluorescent signal from the tweezed particle on and off while maintaining an equivalent tweezing action. Femtosecond lasers can thus be used for optical tweezing and simultaneously utilized to induce nonlinear multi-photon processes such as two-photon excitation or even photoporation.

Photoporation and cell transfection using a violet diode laser

The introduction and subsequent expression of foreign DNA inside living mammalian cells (transfection) is achieved by photoporation with a violet diode laser. We direct a compact 405 nm laser diode source into an inverted optical microscope configuration and expose cells to 0.3 mW for 40 ms. The localized optical power density of ~1200 MW/m2 is six orders of magnitude lower than that used in femtosecond photoporation (~104 TW/m2). The beam perforates the cell plasma membrane to allow uptake of plasmid DNA containing an antibiotic resistant gene as well as the green fluorescent protein (GFP) gene. Successfully transfected cells then expand into clonal groups which are used to create stable cell lines. The use of the violet diode laser offers a new and simple poration technique compatible with standard microscopes and is the simplest method of laser-assisted cell poration reported to date.

Highly efficient blue-light generation from a compact, diode-pumped femtosecond laser by use of a periodically poled KTP waveguide crystal

We present a simplified, potentially portable, and highly efficient blue-light source from a periodically poled KTP waveguide crystal with a compact femtosecond Cr:LiSAF laser. This light source generates 5.6 mW of blue average output power at 424 nm with 27 mW of incident fundamental in a single-pass extracavity arrangement at room temperature. The overall system efficiency of electrical power to blue light is 0.5%, and the internal second-harmonic generation conversion efficiency is as high as 37%. The slope efficiency of 5.5% pJ(-1) at low pulse energies is, to our knowledge, the highest slope efficiency yet reported for frequency conversion into the blue spectral region.

Optically guided neuronal growth at near infrared wavelengths

Recent work has indicated the potential of light to modify the growth of neuronal cells. The two reported studies however, were performed on two independent optical set-ups and on differing cell-types at different temperatures and at different wavelengths. Therefore, it is unknown whether there is a bias for this effect to a particular wavelength which would have implications for the mechanisms for this phenomenon. Localized changes in heat have been suggested as a possible mechanism for this process, but as yet there is no direct experimental evidence to support or discount this hypothesis. In this paper, we report the first direct comparison on one cell type, of this process at two near infra-red wavelengths: 780 nm and 1064 nm using exactly the same beam shape. We show that light at both wavelengths is equally effective in initiating this process. We also directly measure the temperature rise caused by each wavelength in water and its absorption in the cellular medium. The recorded temperature rises are insufficient to change the rate of actin polymerization.

Simplified cavity designs for efficient and compact femtosecond Cr: LiSAF lasers

Abstract We describe efficient, compact femtosecond Cr:LiSAF lasers with a reduced component count that combine greater simplicity with improved performance. We observe transform-limited pulses as short as 136 fs centered on 859 nm at a 470 MHz repetition rate. 20 mW of average output power has been achieved for less than 100 mW of incident diode-laser pump power – an optical-to-optical conversion efficiency of over 20%. We have demonstrated an entirely portable, self-contained, battery-powered version of this laser on a 22×28 cm 2 breadboard, with an electrical-to-optical efficiency of almost 4%. Using four pump laser diodes, we have also achieved operational regimes providing either gigahertz repetition rates or kilowatt peak powers.

Highly compact and efficient femtosecond Cr:LiSAF lasers

Methods for developing more compact femtosecond Cr:LiSAF laser sources are examined. By careful modeling of the low threshold performance and intracavity dispersion characteristics of these cavities, a highly asymmetric z-cavity design with a single prism for dispersion compensation is developed. Transform-limited pulses as short as 113 fs and modelocked output powers up to 20 mW are demonstrated for less than 110 mW of laser-diode pump power. The complete laser system (including the laser diode pump system and drivers) has a footprint of 21.5 /spl times/ 28 cm/sup 2/, about the size of a sheet of US letter or A4 paper.

Second-harmonic generation with focused beams under conditions of large group-velocity mismatch

We present a theoretical model that describes the focusing conditions for second-harmonic generation (SHG) of focused femtosecond pulses as a function of group-velocity mismatch (GVM), with direct application to efficient SHG using a “thick” nonlinear crystal. We observe a direct dependence of the optimal focusing ratio, L/b, on the strength of group-velocity mismatch. Our model also describes the temporal duration of the second-harmonic pulses under these conditions as well as the change in optimal phase mismatch. The theoretical results are compared with an experiment for SHG with focused femtosecond pulses in a “thick” crystal of KNbO3.

Efficient, high repetition-rate femtosecond blue source using a compact Cr:LiSAF laser

We present a practical route to designing a portable femtosecond blue light source that is rugged, compact and battery-powered. An opticaloptical second-harmonic generation (SHG) efficiency of 30% is reported using a diode-pumped, femtosecond Cr:LiSAF laser requiring only ~1.2W of electrical drive. 12mW of blue average power is generated using a 3mm KNbO3 crystal in a simple, single-pass extracavity geometry. The corresponding electrical-blue efficiency of 1% is, to our knowledge, the highest reported efficiency of any femtosecond blue source. Despite conditions of large group velocity mismatch, we show that the temporally-broadened blue pulses remain well within the femtosecond regime, at ~540fs.