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In Figure 1, beams of light are refracted by the particle where the beam intensity increases towards the central beam axis. As light is refracted by the particle, the change in momentum of the beam exerts an opposite force on the particle through which it is propagating. This change in momentum is greater for beam components closer to the axis - causing an off-axis particle to be drawn towards the central axis. The scattering force is due to scattering of light within the particle and is balanced by the net loss of momentum by the refracted beams in the forward direction which tends to trap the particle close to the beam focus.
History of optical tweezers
The first reference to the use of light for positional trapping of particles by light is attributed to Ashkin1 as a researcher in Bell Laboratories in Holmdel, New Jersey and where a counter propagating beam structure was described. With development of techniques, especially of resolution of trapped beams, Ashkin and Dziedzic2 reported the application of single beam gradient traps to control the position of tobacco mosaic viruses and also live bacteria. Early work also described3,4 the safe trapping of a range of biological entities including human red blood cells and various protozoa and where also the orientation of bacteria within such fields could be controlled.
A question uppermost in the minds of investigators was to verify that the intensities to which the cells are exposed do not result in cellular damage. This can be considered to be a function of wavelength, power densities involved and length of time of potential exposure. A commonly used laser for such work is the Ti:sapphire laser with a centre wavelength of around 780nm and where the photon energy is considered too low to initiate cellular damage.
Optical cell transfection
The technique of cell transfection is essentially that of manipulation of cell structure by insertion of foreign DNA material. Prior to the use of lasers, convention techniques had included the use of chemicals to stress cell membranes and also those of direct physical insertion through use of ultra miniature probes, though rates of successful cell transfection associated with such conventional methods were low. The first description of 'optical transfection' is attributed to Tsukakoshi5 where a frequency tripled Nd:YAG laser of wavelength 355nm and 5ns pulse duration briefly opened an aperture of around 0.3 microns in cell walls - allowing DNA material to enter the cell structure. This allowed rates of successful transfection to be increased by around a factor of 10. A useful review by Stevenson6 describes a range of techniques that have been employed, and confirms that the corresponding effectiveness is highly influenced by the pulse duration. For pulse durations longer than nanoseconds, associated mechanisms include heating and photochemical effects. For pulse durations of the order of nanoseconds heating and thermoelastic stress are considered to be involved and where also adjacent cells can be subject to shock waves. In the range of femto second pulses, energy is increasingly translated into generation of highly localised plasmas which open up the cell membrane and with reduced collateral damage to adjacent cells. Current techniques of optical transfection as described by Niioka7 use typically a wavelength of 800nm and with a pulse duration of less than 200 femto seconds and where transfection apertures are usually in the range 0.3 microns to 1.0 microns on the cell membrane surface. Laser modes have also been developed for the use of a single wavelength which can be operated in a continuous mode for 'trapping' but where in femto second-pulsed mode the laser can dissect cell walls to allow removal or insertion of sub components of cell metabolism (organelles).
Optical stretching
One application of optical containment technology is that of 'optical stretching' where a cell is manipulated at a point of focus with light forces pulling on the cell in opposite directions. This allows estimations to be made of the structure of cytoskeleton of cells and which can be used to distinguish between normal and cancerous cells. Automated systems are being developed to achieve high rates of cell identification. The technique of optical stretching is described by Schinkinger8 where modelling of cell stretching dynamics with inner cell configuration identifies the role of optical stretching as a means of cell characterisation even within small cell populations. While this technique is primarily used in applications relating to monitoring cell types within established cell lines, this technique has potential as an active diagnostic technique across a broad range of disease presentations.
An example of this is presented within research into malaria, where the progress of infection within a specific red blood cell can be monitored by its stiffness characteristics as described by Mauritz9 in association with a range of advanced microscopy techniques. In Figure 2 cells pass along a capillary between two counter propagating laser beams and their elongation with time is measured as they pass into the focal point of the optical 'trap'. This technique has the potential to be used as a screening technique for early detection of active malaria infection and where infected cells become progressively more rigid. In addition, manipulation of such parasites with optical tweezers allows investigation of adhesion sites of the malaria parasite Plasmodium sporozoite to better understand mechanisms of cell infection10 and also to determine the forces exerted upon uninfected cells during the process of infection.
The technique of optical tweezers can also be used to rotate selected cells through specific angular rotations as part of specialist imaging procedures. Such techniques also allow measurement of stretching forces to measure forces between interacting molecules and also the forces of insertion which are required for viruses to infect cells. Such information provides another dimension for understanding mechanical forces which are associated with a range of biological processes.
Holographic optical tweezers
In holographic optical tweezers, a single laser beam can be dynamically configured to establish and array of trapping beams - each of which can be dynamically reconfigured to steer multiple objects in real time according to pre-planned trajectories. Padgett and Leonardo at the Glasgow Optics Group describe the applications of such 'holographic' optical tweezers.11 The Glasgow Optics group is headed by Miles Padgett, Professor of Optics in the Department of Physics and Astronomy at the University of Glasgow and where a key focus of the group is harnessing the effects associated with the angular momentum of light.
The optical spanner
A particular line of development in the Glasgow Optics Group has been the development of light beams using specialist modes of orbital angular momentum - so called Laguerre-Gaussian (LG) modes - where beam powers of optical tweezers using such modes can be significantly reduced compared with conventional modes. Orbital angular momentum has also been demonstrated to function like an 'optical spanner'12 where light can induce objects to spin as the angular momentum is communicated to the target site. Many key molecular interactions are considered to involve rotational force interaction as molecules 'screw' into each other.
Single molecule biophysics
Physicists are engaging with life science researchers to investigate intimate life science interactions at the level of individual molecules in an emerging discipline termed 'Single Molecule Biophysics' and which is an overlap of Physics and Biology. Techniques for investigation of 'Single Molecule Biophysics' currently include a broad range of modalities.
One specific technique, that of optical tweezers has been applied under conditions of high sensitivity and stability at Stanford University13 to investigate, for example, interactions of RNA polymerase traversing a strand of DNA and reading base pair configurations at specific positions. In this technique the position of RNA molecules can be tracked in real time showing 'pauses' derived from multiples of base pair separation distances. Thus while science has previously decoded the physical structures of the molecules of 'life' - they can now observe the mechanisms at work.
Equipment
A feature of the field is that a wide range of scientific systems supporting optical manipulation techniques such as optical tweezers is now commercially available. This allows shortening of research processes where intricate laboratory systems can be rapidly installed rather than requiring development over extended periods. Figure 3 shows Eliot Scientific's E3500 computer-controlled multiple-trap optical tweezer system with QPD force measurement facility using two fibre-coupled lasers - one for trapping and one for the probe beam.
Conclusion
'Optical tweezer' technology has emerged as a general purpose tool with which to investigate and study and broad range of biological processes - initially at the cellular level and now also at the molecular level. Such techniques are now available for further developments in research within ophthalmology. The emergence of 'Single Molecule Biophysics' using techniques such as optical tweezers makes possible new lines of investigation in relation to complex processes of molecular interaction and which is often a key strand within ophthalmic research. ?
References
1 Ashkin A. Acceleration and Trapping of Particles by Radiation Pressure, Phys Rev Lett, 1970; 24:156-159.
2 Ashkin A, Dziedzic JM. Optical trapping and manipulation of viruses and bacteria. Science, 1987; 235(4795):1517-20.
3 Ashkin A, Dziedzic JM, Yamane T. Optical trapping and manipulation of single cells using infrared laser beams. Nature, 1987; 330(6150):769-71.
4 Ando J, Bautista G, Smith N, et al. Optical trapping and surgery of living yeast cells using a single laser. Rev Sci Instrum, 2008; 79(10):103705.
5 Tsukakoshi M, Kurata S, NomiyaY et al, A Novel Method of DNA Transfection by Laser Microbeam Cell Surgery. Applied Physics B-Photophysics and Laser Chemistry, 1984; 35(3):135-140.
6 Stevenson DJ, Gunn-Moore FJ, Campbell P, et al. Single cell optical transfection. JR Soc Interface, 2010; 7(47):863-71.
7 Niioka H, Smith NI, Fujita K, et al. Femtosecond laser nano-ablation in fixed and nonfixed cultured cells. Opt Express, 2008; 16(19):14476-14495.
8 Schinkinger S, Wottawah F, Travis K, Lincoln B, Guck J. Feeling for cells with light. Proc SPIE vol 5514, Optical Trapping and Optical Micromanipulation, 170-178 (October 18, 2004); doi:10.1117/12.556795.
9 Mauritz JM, Esposito A, Tiffert T, et al. Biophotonic techniques for the study of malaria-infected red blood cells. Med Biol Eng Comput, 2010; 48(10):1055-63.
10 Hegge S, Uhrig K, Streichfuss M, et al. Direct manipulation of malaria parasites with optical tweezers reveals distinct functions of Plasmodium surface proteins. ACS Nano, 2012; 6(6):4648-62.
11 Padgett M and Di Leonardo R. Holographic optical tweezers and their relevance to lab on chip devices. Lab Chip, 2011; 11: 1196-1205.
12 Padgett MJ, and Allen L. The angular momentum of light: optical spanners and the rotational frequency shift. Optical and Quantum Electronics, 1999; 31(1):1-12.
13 Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, et al. Direct observation of base-pair stepping by RNA polymerase. Nature, 2005; 438(7067):460-5.
? Dr Douglas Clarkson is development and quality manager at the department of clinical physics and bio-engineering, Coventy and Warwickshire University Hospital Trust
