Tanya K. Lake

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.

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.

Optically guided neuronal growth at near-infrared wavelengths

Recent work has indicated the potential of light to guide the growth cones of neuronal cells using a Ti:Sapphire laser at 800 nm (Ehrlicher et al, PNAS, 2002). We have developed an optical set-up that has allowed, for the first time, the direct comparison of this process at near infrared wavelengths. A high number of growth cones were studied in order to provide a detailed statistical analysis. Actively extending growth cones of the neuroblastoma cell-line, NG108, can be guided at not only 780 nm, but also at 1064 nm. These wavelengths are an appropriate choice for guidance experiments, as wavelengths in the visible spectrum and UV are highly absorbing by cells and lead to death by phototoxicity and thermal stress. At 780 nm, 47% of actively extending growth cones were found to turn towards the focused incident light by at least 30° (n=32 growth cones). At 1064 nm, 61% of cells were successfully guided (n=31 growth cones). This suggests that the light detection mechanism within the cell is not due a single protein with a defined activity wavelength as occurs for example with the photoreceptor family of opsin proteins in the mammalian eye. We present two novel mechanisms of light induced neuronal guidance which are not related to temperature increases, or optical tweezing of the growth cone. We are also now identifying the signaling pathways that mediate this phenomenon.

Optical transfection of mammalian cells

The introduction of naked DNA or other membrane impermeable substances into a cell (transfection) is a ubiquitous problem in cell biology. This problem is particularly challenging when it is desired to load membrane impermeable substances into specific cells, as most transfection technologies (such as liposomal transfection) are based on treating a global population of cells. The technique of optical transfection, using a focused laser to open a small transient hole in the membrane of a biological cell (photoporation) to load membrane impermeable DNA into it, allows individual cells to be targeted for transfection, while leaving neighbouring cells unaffected. Unlike other techniques used to perform single cell transfection, such as microinjection, optical transfection can be performed in an entirely closed system, thereby maintaining sterility of the sample during treatment. Here, we are investigating the introduction and subsequent expression of foreign DNA into living mammalian cells by laser-assisted photoporation with a femtosecond-pulsed titanium sapphire laser at 800 nm, in cells that are adherent.

Microlensed red and violet diode lasers in an extended cavity geometry

We examine the behavior of two microlensed diode lasers at 413 and 661 nm placed in both free-running and extended cavity geometries. The 25 mW free-running 413 nm diode shows current tuning over 40 GHz and the 50 mW, 661 nm diode shows tuning of 55 GHz. In extended cavity the 413 nm diode coarsely tunes 5 nm and finely tunes 6 GHz, with a linewidth of 4 MHz. The 661 nm diode coarsely tunes 11 nm with fine tuning of 6 GHz. Beam profile ratios for the 413 and 661 nm microlensed diodes are 1:1.06 and 1:1.10 respectively.

Simultaneous optical tweezing and laser-induced fluorescence of DAPI stained chromosomes

In this paper, we describe methods to increase the functionality of conventional optical tweezer techniques through the use of a 410 nm laser diode to induce fluorescence in biological samples. For the first time, to our knowledge, we demonstrate the use of a violet diode laser to induce fluorescence in particles, which are simultaneously tweezed by an infrared laser. Using this technique we demonstrate the sorting of fluorescent polymer spheres and DAPI-stained chromosomes.

Transfection of cells using a violet diode laser for photoporation

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 tightly focused laser beam (with a localised focal volume of ~10-19 m3 ) 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

Sum frequency mixing of microlensed diode lasers for spectroscopy

UV power of 50.3 nW is obtained using two microlensed diode lasers in a single pass geometry, through sum-frequency mixing in a non-linear BBO crystal. The 413 nm microlensed diode laser is used in external cavity and the 662 nm microlensed diode laser as a free running source. The narrow linewidth and broad tuning range create a tool readily compliant with the stringent requirements of laser spectroscopy.

Compact and efficient single-frequency Nd : YVO/sub 4/ laser with variable longitudinal-mode discrimination

We show for the first time that the longitudinal-mode discrimination in a birefringently filtered laser can be tuned through the variation of the waveplating action of the gain crystal. In this way, the laser can be optimized for either high intermodal discrimination or for frequency tuning with reduced output power rolloff. Up to 760 mW of single-frequency 1064-nm output is obtained from a compact diode-pumped source that can be frequency chirped over 6.5 GHz.

The effect of gain crystal temperature on a miniature single frequency Nd:YVO/sub 4/ laser

Summary form only given. Microchip lasers represent highly efficient sources of laser radiation and are easily mass produced at low cost. However, it is difficult to operate them on a single frequency at output powers greater than about 150 mW. Hence, laser geometries that maintain the simplicity exhibited by microchip lasers yet which can be operated on a single frequency at higher output powers are of great interest. In the paper we demonstrate the potential of a birefringent filter, consisting of a Brewster plate and a birefringent crystal, as a frequency selective element in a micro-laser which has additionally, a birefringent gain crystal. Single frequency output powers greater than 760 mW have been obtained at 1064 nm for 2 W of diode laser pump power.