Christian T. A. Brown

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.

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.

Targeted optical injection of gold nanoparticles into single mammalian cells

We present an all optical technique for the targeted delivery of single 100 nm diameter gold nanoparticles into a specified region of the interior of an individual mammalian cell through a combination of optical tweezing and optical injection. The internalisation of the nanoparticle is verified by confocal laser scanning microscopy and confocal laser scanning reflectance microscopy. This represents the first time that nano sized particles have been tweezed and optically injected into mammalian cells using only light, and provides a novel methodology for internalising nanosphere based biosensors within specific intracellular regions of a mammalian cell.

Optical Separation of Cells on Potential Energy Landscapes: Enhancement With Dielectric Tagging

We review the emergent techniques of microfluidic sorting of colloidal and cellular samples using optical forces. We distinguish between what we term as passive and active forms of particle sorting where we can sort either with the use of a fluorescent marker (active) or based on physical attributes alone (passive). We then examine cell sorting with optical potential landscapes such as a Bessel light beam and a multibeam interference pattern. For both forms of optical potential energy landscape, we further present the possibility of enhancing the optical sorting process by tagging dielectric microspheres onto the cells. The results suggest that the methodology of tagging can enhance the sorting of cells as they subsequently respond more strongly to an applied optical field or potential energy landscape. This technique presents a simple method to enhance the sorting process.

Fundamentally mode-locked, femtosecond waveguide oscillators with multi-gigahertz repetition frequencies up to 15 GHz

We demonstrate passively mode-locked Yb(3+)-doped glass waveguide lasers in a quasi-monolithic configuration with a maximum pulse repetition frequency up to 15.2 GHz. A semiconductor saturable absorber mirror (SESAM) is used to achieve stable mode-locking around 1050 nm with pulse durations as short as 811 fs and an average power up to 27 mW. Different waveguide samples are also employed to deliver pulses with repetition rates of 4.9 GHz, 10.4 GHz and 12 GHz with an average power of 32 mW, 60 mW and 45 mW, respectively. The group velocity dispersion control in the cavity is provided by changing the gap between the SESAM and the waveguide end-face to facilitate a soliton mode-locking regime.

Compact laser-diode-based femtosecond sources

This paper describes the development of compact femtosecond laser systems that are capable of being directly pumped by laser diodes or are based directly on laser diodes. The paper demonstrates the latest results in a highly efficient vibronic based gain medium and a diode-pumped Yb:KYW laser is reported that has a wall plug efficiency >14%. A Cr4+:YAG oscillator is described that generates transform-limited pulses of 81 fs duration at a pulse repetition frequency of >4 GHz. The development of Cr3+:LiSAF lasers that can be operated using power supplies based on batteries is briefly discussed. We also present a summary of work being carried out on the generation of fs-pulses from laser diodes and discuss the important issues in this area. Finally, we outline results obtained on the generation of pulses as short as 550 fs directly from a two-section quantum dot laser without any external pulse compression.

Imaging in optical micromanipulation using two-photon excitation

Recent studies have realized optical micromanipulation of extended two- and three-dimensional structures and optical binding. In many recent experiments the optical interaction between particles and their light scattering has played a key role. We use fluorescein dye within the sample medium and a femtosecond laser for optical micromanipulation. By the process of two-photon excitation we can directly observe how the light behaves during refraction and reflection within a sample of optically guided microspheres. We directly visualize the reconstruction of the Bessel light beam by excitation of two-photon fluorescence when used as an optical guide for microscopic particles dispersed within this dyed medium. This technique may assist in the visualization of optical scattering and refraction in micromanipulation and the creation of large optically bound matter crystals.

Discrimination of normal from pre-malignant cervical tissue by Raman mapping of de-paraffinized histological tissue sections.

The authors present Raman cluster mapping of de-paraffinized normal cervical tissue and demonstrate the ability of this approach to differentiate between normal squamous epithelium and cervical intraepithelial neoplasia (CIN). Multivariate analysis was performed by hierarchical cluster analysis (HCA) of the Raman spectra associated with the different tissue types and Raman maps were generated using the resultant clusters. Using normal cervical tissue, squamous epithelium and the epithelial-stromal interface, a muscular artery and endocervical glands were successfully mapped. Analysis of a tissue section containing a cervical intraepithelial neoplasia (CIN) grade 2 lesion adjacent to normal squamous epithelium demonstrated that the CIN lesion clustered predominantly with the basal epithelial cells of normal epithelium and allowed visual discrimination of these areas using the Raman map. These findings suggest that Raman mapping has the potential to provide images that are useful for disease diagnosis. In particular, the discrimination between normal cervical squamous epithelium and CIN is of relevance to cervical screening pathology.

Monte Carlo simulations for optimal light delivery in photodynamic therapy of non-melanoma skin cancer

The choice of light source is important for the efficacy of photodynamic therapy (PDT) of non-melanoma skin cancer. We simulated the photodynamic dose (PDD) delivered to a tumour during PDT using theoretical radiation transfer simulations performed via our 3D Monte Carlo radiation transfer (MCRT) model for a range of light sources with light doses up to 75 J cm(-2). The PDD delivered following superficial irradiation from (A) non-laser light sources, (B) monochromatic light, (C) alternate beam diameters and (D) re-positioning of the tumour within the tissue was computed. (A) The final PDD deposited to the tumour at a depth of 2 mm by the Paterson light source was 2.75, 2.50 and 1.04 times greater than the Waldmann 1200, Photocure and Aktilite, respectively. (B) Tumour necrosis occurred at a depth of 2.23 mm and increased to 3.81 mm for wavelengths 405 and 630 nm, respectively

Monte Carlo modelling of daylight activated photodynamic therapy

The treatment of superficial skin lesions via daylight activated photodynamic therapy (PDT) has been explored theoretically with three dimensional (3D) Monte Carlo radiation transfer simulations. For similar parameters and conditions, daylight activated PDT was compared to conventional PDT using a commercially available light source. Under reasonable assumptions for the optical properties of the tissue, protoporphyrin IX (PpIX) concentration and a treatment dose of 75 J cm(-2), it was found that during a clear summer day an effective treatment depth of over 2 mm can be achieved after 30 min of daylight illumination at a latitude of 56 degrees North. The same light dose would require 2.5 h of daylight illumination during an overcast summer day where a treatment depth of about 2 mm can be achieved. For conventional PDT the developed model suggests that 15 min of illumination is required to deliver a light dose of 75 J cm(-2), which would result in an effective treatment depth of about 3 mm. The model developed here allows for the determination of photo-toxicity in skin tissue as a function of depth for different weather conditions as well as for conventional light sources. Our theoretical investigation supports clinical studies and shows that daylight activated PDT has the potential for treating superficial skin lesions during different weather conditions.