Fibre based cellular transfection.

Optically assisted transfection is emerging as a powerful and versatile method for the delivery of foreign therapeutic agents to cells at will. In particular the use of ultrashort pulse lasers has proved an important route to transiently permeating the cell membrane through a multiphoton process. Though optical transfection has been gaining wider usage to date, all incarnations of this technique have employed free space light beams. In this paper we demonstrate the first system to use fibre delivery for the optical transfection of cells. We engineer a standard optical fibre to generate an axicon tip with an enhanced intensity of the remote output field that delivers ultrashort (~ 800 fs) pulses without requiring the fibre to be placed in very close proximity to the cell sample. A theoretical model is also developed in order to predict the light propagation from axicon tipped and bare fibres, in both air and water environments. The model proves to be in good agreement with the experimental findings and can be used to establish the optimum fibre parameters for successful cellular transfection. We readily obtain efficiencies of up to 57 % which are comparable with free space transfection. This advance paves the way for optical transfection of tissue samples and endoscopic embodiments of this technique.

Long-range one-dimensional longitudinal optical binding.

We create extended longitudinally optically bound chains of microparticles with the use of counterpropagating "nondiffracting" light fields, the so-called Bessel beams. The beam homogeneity and extended propagation range allow the creation of 200 microm long chains of organized microparticles. We observe short-range multistability within a single chain and long-range multistability between several distinct chains. Our observations are supported by theoretical results of the coupled dipole method.

Optical deflection and sorting of microparticles in a near-field optical geometry.

Near-field optical micromanipulation permits new possibilities for controlled motion of trapped objects. In this work, we report an original geometry for optically deflecting and sorting micro-objects employing a total internal reflection microscope system. A small beam of laser light is delivered off-axis through a total internal reflection objective which creates an elongated evanescent illumination of light at a glass/water interface. Asymmetrical gradient and scattering forces from this light field are seen to deflect and sort polystyrene microparticles within a fluid flow. The speed of the deflected objects is dependent upon their intrinsic properties. We present a finite element method to calculate the optical forces for the evanescent waves. The numerical simulations are in good qualitative agreement with the experimental observations and elucidate features of the particle trajectory. In the size range of 1 microm to 5 microm in diameter, polystyrene spheres were found to be guided on average 2.9 +/- 0.7 faster than silica spheres. The velocity increased by 3.0 +/- 0.5 microms(-1) per microm increase in diameter for polystyrene spheres and 0.7 +/- 0.2 microms(-1) per microm for silica. We employ this size dependence for performing passive optical sorting within a microfluidic chip and is demonstrated in the accompanying video.

Dual beam fibre trap for Raman micro-spectroscopy of single cells.

Raman spectroscopy permits acquisition of molecular signatures from both cellular and sub-cellular samples. When combined with optical trapping we may interrogate an isolated cell reducing extraneous signals from the local environment. To date, experimental configurations have employed combinations of the single beam optical tweezers trap and Raman spectroscopy, using either the same beam or separate beams for Raman interrogation and trapping. A key problem in optical tweezers is the ability to hold and manoeuvre large cells. In this paper, we use a dual beam fibre trap to hold and manoeuvre cells combined with an orthogonally placed objective to record Raman spectra. The dual beam trap, due to its divergent light fields, offers an as yet unexploited ability to hold and move large cellular objects with reduced prospects of photodamage. We additionally show how this system permits us to move large primary human keratinocytes (approximately 30 microns in diameter), such that we may record Raman spectra from local parts of a trapped cell with ease. Finally, we develop a rudimentary microfluidic system used to generate a flow of cells. Using our dual beam trap, combined with this flow system, we hold and acquire Raman spectra from individual cells chosen from a sample of HL60 human promyelocytic leukemia cells.

Interference from multiple trapped colloids in an optical vortex beam.

Laguerre-Gaussian (LG) beams are important in optical micromanipulation. We show that optically trapped microparticles within a monochromatic LG beam may lead to the formation of unique intensity patterns in the far field due to multiple interference of the forward scattered light from each particle. Trapped colloids create far field interference that exhibits distinct spiral wave patterns that are directly correlated to the helicity of the LG beam. Using two trapped particles, we demonstrate the first microscopic version of a Young's slits type experiment and detect the azimuthal phase variation around the LG beam circumference. This novel technique may be implemented to study the relative phase and spatial coherence of two points in trapping light fields with arbitrary wavefronts.

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.

Optical guiding of microscopic particles in femtosecond and continuous wave Bessel light beams.

Optical guiding of microscopic particles is studied and compared for femtosecond and continuous wave Bessel light beams. We confirm that optical guiding is an average power effect and observe no difference in the guiding velocities for non-fluorescing polymer microspheres. Additionally, we observe second harmonic generation of guided KTP particles. This observation opens up the prospect for optical identification of guided cells for sorting purposes.

Observation of the transfer of the local angular momentum density of a multiringed light beam to an optically trapped particle.

We observe the spinning and orbital motion of a microscopic particle trapped within a multiringed light beam that arises from the transfer of the spin and orbital components of the light's angular momentum. The two rotation rates are measured as a function of the distance between the particle and the axis of the trapping beam. The radial dependence of these observations is found to be in close agreement with the accepted theory.

Interfering Bessel beams for optical micromanipulation.

We examine the properties of interfering high-order Bessel beams. We implement an experimental setup that allows us to realize these interferograms, using interfering Laguerre-Gaussian beams and an axicon. We demonstrate the use of such beams for controlled rotation of microscopic particles in optical tweezers and rotators. The self-healing properties of interfering Bessel beams allow the simultaneous manipulation and rotation of particles in spatially separated sample cells.

Simultaneous micromanipulation in multiple planes using a self-reconstructing light beam.

Optical tweezers are commonly used for manipulating microscopic particles, with applications in cell manipulation, colloid research, manipulation of micromachines and studies of the properties of light beams. Such tweezers work by the transfer of momentum from a tightly focused laser to the particle, which refracts and scatters the light and distorts the profile of the beam. The forces produced by this process cause the particle to be trapped near the beam focus. Conventional tweezers use gaussian light beams, which cannot trap particles in multiple locations more than a few micrometres apart in the axial direction, because of beam distortion by the particle and subsequent strong divergence from the focal plane. Bessel beams, however, do not diverge and, furthermore, if part of the beam is obstructed or distorted the beam reconstructs itself after a characteristic propagation distance. Here we show how this reconstructive property may be utilized within optical tweezers to trap particles in multiple, spatially separated sample cells with a single beam. Owing to the diffractionless nature of the Bessel beam, secondary trapped particles can reside in a second sample cell far removed ( approximately 3 mm) from the first cell. Such tweezers could be used for the simultaneous study of identically prepared ensembles of colloids and biological matter, and potentially offer enhanced control of 'lab-on-a-chip' and optically driven microstructures.

Analysis of ferroelectric 180 degrees -domain structures in BaTiO(3) by use of second-harmonic scattering.

We present a method of determining the distribution of sizes of ferroelectric 180 degrees domains in BaTiO(3), using second-harmonic scattering. We find that the domains are rods with approximately square cross sections that extend from the -c to the +c face of the crystal, with widths ranging from 2 to 150 mum.