Lynn Paterson

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.

Light-induced cell separation in a tailored optical landscape

We demonstrate passive optical sorting of cell populations in the absence of any externally driven fluid flow. Specifically, we report the movement of erythrocytes and lymphocytes in an optical landscape, consisting of a circularly symmetric light pattern created by a Bessel light beam. These distinct cell populations move, spontaneously and differentially, across the underlying periodic optical landscape. Thus, we were able to separate lymphocytes from a mixed population of cells containing erythrocytes and then collect the lymphocytes in a microcapillary reservoir. We also demonstrate an enhanced form of this separation that exploits the polarizability of silica microspheres by attaching spheres coated with antibodies to cell surface markers to a subpopulation of lymphocytes. These techniques may be applied using standard laboratory apparatus.

Trapping and manipulation of low-index particles in a two-dimensional interferometric optical trap

We demonstrate optical trapping and manipulation of low-index spheres in two dimensions, using the pattern produced by two interfering plane waves. This technique shows, for what is believed to be the first time, alignment of an array of hollow spheres and simultaneous manipulation of high- and low-index particles in the horizontal plane. Furthermore, rodlike particles (up to 30microm in length) are manipulated simultaneously with the low-index particles. This technique offers a practical method for manipulating bubbles, low-index droplets, or rodlike biological samples.

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.

Revolving interference patterns for the rotation of optically trapped particles

Abstract Optically trapped objects are rotated controllably in the interference pattern between a Laguerre–Gaussian (LG) beam and a Gaussian beam. In this work the interference pattern is analysed and its properties as it propagates are modelled, showing the important role played by the Guoy-phase of the two interfering beams. An analysis of producing controlled rotation of the interference pattern using a glass plate is presented demonstrating the ease with which the rotation can be controlled.

Multiplane imaging and three dimensional nanoscale particle tracking in biological microscopy

A conventional microscope produces a sharp image from just a single object-plane. This is often a limitation, notably in cell biology. We present a microscope attachment which records sharp images from several object-planes simultaneously. The key concept is to introduce a distorted diffraction grating into the optical system, establishing an order-dependent focussing power in order to generate several images, each arising from a different object-plane. We exploit this multiplane imaging not just for bio-imaging but also for nano-particle tracking, achieving approximately 10 nm z position resolution by parameterising the images with an image sharpness metric.

A 3D mammalian cell separator biochip

The dissimilar cytoskeletal architecture in diverse cell types induces a difference in their deformability that presents a viable approach to separate cells in a non-invasive manner. We report on the design and fabrication of a robust and scalable device capable of separating a heterogeneous population of cells with variable degree of deformability into enriched populations with deformability above a certain threshold. The three dimensional device was fabricated in fused silica by femtosecond laser direct writing combined with selective chemical etching. The separator device was evaluated using promyelocytic HL60 cells. Using flow rates as large as 167 μL min(-1), throughputs of up to 2800 cells min(-1) were achieved at the device output. A fluorescence-activated cell sorting (FACS) viability analysis on the cells revealed 81% of the population maintain cellular integrity after passage through the device.

Quantum Dot‐Based Thermal Spectroscopy and Imaging of Optically Trapped Microspheres and Single Cells

Laser-induced thermal effects in optically trapped microspheres and single cells are investigated by quantum dot luminescence thermometry. Thermal spectroscopy has revealed a non-localized temperature distribution around the trap that extends over tens of micrometers, in agreement with previous theoretical models besides identifying water absorption as the most important heating source. The 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 is corroborated by a simultaneous analysis of the spectral dependence of cellular heating and damage in human lymphocytes during optical trapping. This quantum dot luminescence thermometry demonstrates that optical trapping with 820 nm laser radiation produces minimum intracellular heating, well below the cytotoxic level (43 °C), thus, avoiding cell damage.

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.

Moving interference patterns created using the angular Doppler-effect

We use the angular Doppler-effect to obtain stable frequency shifts from below one Hertz to hundreds of Hertz in the optical domain, constituting a control of 1 part in 1014. For the first time, we use these very small frequency shifts to create continuous motion in interference patterns including the scanning of linear fringe patterns and the rotation of the interference pattern formed from a Laguerre-Gaussian beam. This enables controlled lateral and rotational movement of trapped particles.