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.
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.
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.
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.
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.
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