A microfluidic device consisting of a 3D network of buried microchannels and integrated waveguides has been fabricated and used to controllably manipulate particles within the micro channels. The channels and waveguides were made using the direct laser writing technique of ultrafast laser inscription, followed by selective chemical etching to fabricate channels.
Particles flowing through the device undergo hydrodynamic flow focusing into a narrow stream within a main channel due to the geometry of the channel network. 3D hydrodynamic focusing performance was validated using polystyrene microspheres, coloured dye and cells by visualizing the focusing within the device. A focusing width of 4 um was achieved, reducing the risk of particles sticking to walls, clogging the channel and ensuring all particles pass the beam. Particles are irradiated by 1064 nm light, in a direction perpendicular to the flow, from the embedded waveguide, causing a lateral displacement of the particle due to the optical scattering force. 5 and 10 micron beads in water were focused to a narrow stream. Lateral displacement was evaluated for 5 different laser powers for particles flowing at a constant velocity >1 mm/s. A linear increase in displacement of the particles with laser power was observed. Bacteria, yeast, microalgae and mammalian cells have been flow-focused and optically manipulated within the device. The device is capable of both passive and active separation of particle species, and the routing of particles to required outlets demonstrates potential for cell sorting.
Silver nanoparticles (Ag-NP) with Surface Enhanced Raman Scattering (SERS) activity were fabricated on a fused silica substrate by ultrafast femtosecond laser photoreduction of a silver salt solution. The SERS effectiveness of the Ag-NP increased with laser writing power and number of scans. SEM images show that the Ag-NP have a more uniform density distribution when using a multi-scan writing technique. A number of different laser parameters were compared, including scan speed, laser power, and number of scans. Overall, it was found that the most effective laser parameters were: 20 µms-1 scan speed, 10 mW laser power and 200 scans. The Ag-NP substrates have been used to detect single bacteria and hold promise to give fast, accurate and specific spectra according to the cell specimen present.
Optical Tweezing is a non-invasive technique that can enable a variety of single cell experiments or cell-cell communication experiments. To date, optical tweezers tend to be based on a high numerical aperture microscope objective to deliver the tweezing light and image the sample, which introduces restrictions in terms of flexibility. A single optical fibre-based probe able to manipulate microparticles independently from the imaging system is demonstrated. The working principle of the probe is based upon two crossed beams that can be used to trap a microparticle in the area where the two beams overlap. The two deflected beams are produced by incorporating fibre-end facet mirrors onto a multicore fibre using a Focused Ion Beam fabrication technique. The light from the two cores overlaps close to the end of the fibre and has been demonstrated to be capable of trapping particles in the area where the beams intersect. By using a multicore fibre instead of separate fibres glued together results in simplified probe manufacture and alignment and offers a smaller probe that is suitable for use in a wider range of applications, including on-chip manipulation
Optical micromanipulation techniques and microfluidic techniques can be used in same platform for manipulating biological samples at single cell level. Novel microfluidic devices with integrated channels and waveguides fabricated using ultrafast laser inscription combined with selective chemical etching can be used to enable sorting and isolation of biological cells. In this paper we report the design and fabrication of a three dimensional chip that can be used to manipulate single cells in principle with a higher throughput than is possible using optical tweezers. The capability of ultrafast laser inscription followed by selective chemical etching to fabricate microstructures and waveguides have been utilised to fabricate the device presented in this paper. The complex three dimensional microfluidic structures within the device allow the injected cell population to focus in a hydrodynamic flow. A 1064 nm cw laser source, coupled to the integrated waveguide, is used to exert radiation pressure on the cells to be manipulated. As the cells in the focussed stream flow past the waveguide, optical scattering force induced by the laser beam pushes the cell from out of the focussed stream to the sheath fluid, which can be then collected at the outlet. Thus cells can be controllably deflected from the focussed flow to the side channel for downstream analysis or culture.
Development of efficient methods for isolation and manipulation of microorganisms is essential to study unidentified and yet-to-be cultured microbes originating from a variety of environments. The discovery of novel microbes and their products have the potential to contribute to the development of new medicines and other industrially important bioactive compounds. In this paper we describe the design, fabrication and validation of an optofluidic device capable of redirecting microbes within a flow using optical forces. The device holds promise to enable the high throughput isolation of single microbes for downstream culture and analysis. Optofluidic devices are widely used in clinical research, cell biology and biomedical engineering as they are capable of performing analytical functions such as controlled transportation, compact and rapid processing of nanolitres to millilitres of clinical or biological samples. We have designed and fabricated a three dimensional optofluidic device to control and manipulate microorganisms within a microfluidic channel. The device was fabricated in fused silica by ultrafast laser inscription (ULI) followed by selective chemical etching. The unique three-dimensional capability of ULI is utilized to integrate microfluidic channels and waveguides within the same substrate. The main microfluidic channel in the device constitutes the path of the sample. Optical waveguides are fabricated at right angles to the main microfluidic channel. The potential of the optical scattering force to control and manipulate microorganisms is discussed in this paper. A 980 nm continuous wave (CW) laser source, coupled to the waveguide, is used to exert radiation pressure on the particle and particle migrations at different flow velocities are recorded. As a first demonstration, device functionality is validated using fluorescent microbeads and initial trials with microalgae are presented.
Laser-induced thermal effects in optically trapped microspheres and single cells have been investigated by Luminescence
Thermometry. Thermal spectroscopy has revealed a non-localized temperature distribution around the trap that extends
over tens of microns, in agreement with previous theoretical models. Solvent absorption has been identified as the key
parameter to determine laser-induced heating, which can be reduced by establishing a continuous fluid flow of the
sample. Our experimental results of thermal loading at a variety of wavelengths reveal that an optimum trapping
wavelength exists for biological applications close to 820 nm. This has been corroborated by a simultaneous analysis of
the spectral dependence of cellular heating and damage in human lymphocytes during optical trapping. Minimum
intracellular heating, well below the cytotoxic level (43 °C), has been demonstrated to occur for optical trapping with 820 nm laser radiation, thus avoiding cell damage.
Recent advances in the field of ultrafast laser inscription provide ample evidence underscoring the
potential of this technique in fabricating novel and previously unthinkable 2D and 3D photonic and
optofluidic platforms enabling current sensor, diagnostics, monitoring and biochemical research to
scale new heights. In addition to meeting the demands for compact, active waveguide devices
designed for diverse applications such as optical metrology, non-linear microscopy and
astrophotonics, this technology facilitates the integration of microfluidics with integrated optics
which creates a powerful technology for the manufacture of custom lab-on-chip devices with
advanced functionality. This paper highlights the capabilities of ultrafast laser inscription in
fabricating novel 3D microfluidic devices aimed for biomedical applications.
A limited range of instruments are available which allow the controlled injection of sub-picolitre volumes;
microfluidic devices and commercially produced mechanical microinjection systems accounting for the majority. We
present an optically controlled microsyringe capable of dispensing femtolitres of liquid. Triple beam optical tweezers are
used to manipulate hollow glass microneedles and also polymer microspheres which were used as 'handles' to assist the
manipulation of microneedles and 'plungers' to dispense liquid from the microneedle.
Standard optical tweezers were used with the addition of a Ronchi ruling (250 lines per inch) mounted in the image
relay telescope. The diffraction pattern generated by the Ronchi ruling produced three optical traps in the sample
chamber. Trap spacing was controlled by translating the ruling along the axis of beam propagation within the image relay
Utilizing the three-beam trap, it was possible to manipulate pulled, borosilicate capillaries (5-150μm in length, 1-10μm
in diameter) both perpendicular and parallel to the axis of the capillary. Rolled SiO/SiO2 microtubes (4μm diameter,
50μm long) were also manipulated, however in this case polymer microspheres were used as 'handles'. In both cases the
microneedles did not align vertically along the propagation axis; an advantage over using a single beam optical trap.
Tweezing a microsphere within a microneedle dispenses femtolitres of liquid from the needle. The force exerted on
microneedles is calculated to be in the order of picoNewtons so may have applications where femtolitre volumes must be
controllably delivered beyond a barrier, such as single cell microinjection.
A continuous flow microfluidic cell separation platform has been designed and fabricated using femtosecond laser
inscription. The device is a scalable and non-invasive cell separation mechanism aimed at separating human embryonic
stem cells from differentiated cells based on the dissimilarities in their cytoskeletal elasticity. Successful demonstration
of the device has been achieved using human leukemia cells the elasticity of which is similar to that of human embryonic
A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam—the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.
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.
Microscopic particles with varying optical properties may be induced to move in different ways when placed on a sculpted optical potential due to differences in shape, size or polarisability. The separation of red blood cells (erythrocytes) and white blood cells (lymphocytes) is achieved in a non-invasive manner and in the absence of any microfluidic systems using a 'non-diffracting' circularly symmetric Bessel beam. The Bessel beam, which consists of a series of concentric rings, each of equal power and of 3.2μm thickness with a spacing of 2μm around a central maximum of 5μm diameter (and is akin to a rod of light as its propagation distance is 3mm), is directed upward into a sample chamber containing blood. Fluctuations in Brownian motion cause cells to escape from individual rings of the Bessel beam and travel towards the beam centre, where the intensity of the rings increases. However, these cells must be able to overcome the potential barrier of each ring which gets larger toward the central maximum. Lymphocytes - spherical in shape and 7μm in diameter (therefore overlapping two rings) - are transported, due to the gradient force of the optical field, to the beam centre where they are guided upwards and form a vertical stack, whereas erythrocytes re-align on their sides in the outer rings and are then guided upwards, because once aligned they cannot escape the potential barrier and 'lock-in' to that ring. The optical power required for optimal sorting in this static sorter which requires no fluid flow is investigated.
Optical binding may arise due to interplay between light scattering and refraction creating equilibrium positions for particles in a self-consistent manner. Binding is observed for the first time in biological cells within a dual beam fiber trap.
Raman Tweezers Micro Spectroscopy has become an important and versatile technique in recent years. The technique is the amalgamation of optical tweezers and traditional Raman spectroscopy. The combination of these two well established techniques has brought key advantages in the studies of many different physical and biological systems from studying drug distribution in cells to measuring the size of aerosol particles. In this paper we present our Raman Tweezers system and discuss its advantages over conventional Raman systems, also discussed in this section is the parameters which effect collection of Raman scattered light using the ability of the optical tweezers to stack micro spheres. Finally we discuss how to extend further the functionality of the Raman tweezers technique by decoupling the trapping and excitation with the use of a fibre optical light force trap.
Optical micro-manipulation has seen a resurgence of interest in recent years which has been due in part to new application areas and the use of tailored forms of light beam. In this paper, experimental observations of fluctuation-driven transport of silica microspheres within a two-dimensional optical potential of circular symmetry are observed. The potential is created by a Bessel light beam. The optical field is tailored to break the symmetry and create a static tilted periodic (washboard) potential. Transitions between locked and running modes may be observed. The running mode manifests itself by rapid accumulation of particles at the beam centre. We discuss what happens with mixtures of particles in such an optical potential.
The path that a mesoscopic polarisable particle takes as it flows through a lattice of intensity maxima and minima (optical lattice) depends crucially upon the degree to which it interacts with the lattice. Two particles of dissimilar size, refractive index or even shape will interact in a different manner with such a lattice. Combining this selective interaction with a guiding mechanism has allowed us to achieve lateral separation of particles by all these properties simply by flowing them through an angled optical lattice. We present such particle separation in a variety of three-dimensional optical lattices discussing the importance of parameters such as flow speed, lattice intensity, lattice constant, lattice angle, maxima interconnectivity and flow chamber design. We also present cell sorting with the separation of erythrocytes from lymphocytes and present our flow chamber fabrication methods.
Laguerre-Gaussian (LG) laser modes (annular shaped modes with helical phase fronts) are used to both manipulate and cut microscopic particles. We use holographically produced LG laser modes to manipulate microscopic bubbles. Interference patterns formed from LG modes of opposite phase helicity are used to create 3D structures and to continuously rotate glass rods. The technique of using and LG beam to create microscopic sections of chromosomes is described.
We demonstrate the use of the angular Doppler effect to obtain continuous motion of interference patterns. A small frequency shift between two beams can create such a moving pattern. By rotating a half wave plate in one arm of an interferometer, frequency shifts in the optical domain from less than 1 Hertz to kHz are achieved. We apply moving interference patterns in an optical tweezers set-up to enable controlled and continuous motion of optically trapped particles and structures.