Building Optical Matter with Binding and Trapping Forces

Jean-Marc Fournier*, Gerben Boer, Guy Delacrétaz, Pierre Jacquot, Johann Rohner, René P. Salathé STI - LOA, EPFL, Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland

ABSTRACT Very high frequency oscillations of intense light fields interact with micron-size dielectric objects to exert dc optical forces that allow polarizable particles to levitate, to be trapped and to be bound. Such optical forces are also suitable to arrange cold atoms in optical lattices. Various assemblages of optical traps, including periodic arrays, can be constructed either with independent lasers, or with a single laser beam split into different parts later recombined by interference, as well as through the use of diffractive elements. These optical-well arrays serve as templates for writing and erasing dynamic two-dimensional and three-dimensional "optical crystals", composed of mono-dispersed polystyrene spheres in water. Subsequently, the crystals become diffractive structures themselves. The association of micro-fluidics and optical trapping allows for the formation of optical traps into micro-channels. This leads to perform microchemistry experiments, such as fluorescence detection, on individual bodies attached to trapped particles. Self-trapping due to the optical binding force relates to the interaction between different dielectric objects located in an electromagnetic field; each one reacts not only to the field of the incident beam, but also to the induced fields radiated coherently by all other particles. Optical binding strongly influences the equilibrium state and the behavior of optical crystals. It must have the potential for creating collective effects. Keywords: Optical trapping, optical binding, optical crystals, optical quasicrystals, microfluidic, diffractive optical elements, interferometry, holography, Talbot trapping, speckle

1. INTRODUCTION Optical traps are widely used by various communities. In atomic physics such optical wells allow the guiding of cold atoms, the trapping of atoms, fermions or bosons, the transportation of Bose-Einstein condensates over very long distances, the building of atom lattices where matter constituting a very diluted gas state is arranged according to crystalline structures as in solids. In biology and in biophysics, optical traps have found many niches of applications, going from encaging many different types of biological objects to measuring extremely weak forces involved in the very essential functions of life, such as mechanisms of muscle contraction, folding, unfolding, and stretching of DNA, study of in-vivo reproduction, just to name a few. There is now a strong push to construct lab-on-a-chip devices in which multiple sets of optical tweezers are coupled with other techniques such as microfluidics to perform single molecule chemistry and to multiply the number of essays performed at once on one or several proteins, or on various cells, or even on vesicles. In this article, we explore several approaches leading to such multiple-trap devices. First, we will look at a set of independent traps generated in a chip containing microfluidic channels, we will measure the stiffness of traps and explore the possibility of capturing fluorescence from trapped bodies. We will also look at interferometric and holographic/diffractive setups allowing the simultaneous production of very high numbers of traps. It will be shown that such large assemblies of traps must be studied by taking into account the whole field, that is to say the coherent addition of the initial trapping field with the fields induced in all trapped objects.

2. MULTIPLE LASER SOURCE TRAPPING. 3D OPTICAL TRAPS IN A MICRO-FLUIDIC SYSTEM

In this section, we present a system that combines four 3D optical traps with a micro-fluidic system. The design of the setup allows fluorescence excitation of the trapped objects. Figure 1 shows a diagram of the whole system. Four fibred * jeanmarc.fournier@epfl.ch

monomode lasers, each of them delivering a maximal optical power of 100 mW at 974 nm, are used as individual sources to create 4 traps. Since the lasers are not phased together, they do not interact and consequently they generate a set of traps which stand alone. Plano-convex lenses collimate the laser beams to a size that matches the 6 mm entrance pupil diameter of the microscope objective (MO). Two sets of adjustable dielectric mirrors fold the beams and redirect them at adequate angles to couple them to the microscope objective (Leica, C-plan, 100 X, NA=1.25, oil immersion) via two additional mirrors. The micro-fluidic silicone chip is placed in an adapted holder on top of the microscope objective. It is positioned with a x-y translation table so that the traps lie within the micro-channel. Along the optical axis direction (z-direction), the traps sit 5 to 10 µm above the bottom of the channel. . As shown in figure 2-a, the four beams form four traps in the front focal plane of the MO. A CCD camera mounted on the video port of the microscope and a white light illumination permit the observation of the trapped objects and the micro-channel through the same MO.

Fig. 2. Schematic side-view of the four traps in a micro-channel. (a) The traps are realized by focusing into a micro-channel the four beams tilted at different incidence angles. _ (b) The fluorescent beads are exited with a slightly focalised excitation laser beam sent through the microscope objective.

Slightly focused fluorescence excitation laser beam

Fluorescent beads

b a

Laser tweezers

Micro-channel

Inverted microscope

Fibred laser diodes at 974 nm

CCD camera

Micro-fluidic system

Fig.1. Schematic top-view of the optical set-up. The four collimated beams are redirected by sets of mirrors and coupled in a high NA objective via the fluorescence port of the inverted microscope. The micro-fluidic chip is installed on top of the objective. A fluorescence excitation laser is coupled into the microscope objective via the side port of the microscope.

Fluorescence excitation laser at 546 nm

The excitation wavelength is provided by a He-Ne laser at 546 nm coupled into the MO via a side port of the microscope. Before entering the microscope, the beam is adequately resized and focused with a two-lens system. Figure 2-b shows how the slightly focused beam illuminates the whole channel and consequently excites fluorescent beads that are routinely trapped. The chip is fabricated by means of a well-known silicone molding technology1. The channel is 150 µm wide and 50 µm deep. The flow through the channels is controlled with a syringe that is actuated with a micrometer screw. The syringe is connected to the chip via a flexible plastic tube. Thanks to the powerful lasers and the high quality focusing, the produced three-dimensional traps are very stiff. Such strong stiffness is required when working with micro-fluidics in order to sustain high flow velocities and bead collisions. The transverse trapping forces of the optical tweezers were previously measured using the viscous drag force technique2. For 2 µm polystyrene beads (Polysciences) immersed in deionized water, we measured that the trapped beads could withstand a maximal translation speed of 800 µm/s, which corresponds, according to the Navier-Stokes equation, to a maximal trapping force of about 15 pN. The experimental setup allows objects of interest flowing into the channel to be caught and fluorescence tests to be performed on them. This is demonstrated in figure 3 where a mixture of fluorescent (∅ 4 µm, Molecular Probes A8858) beads and non-fluorescent beads (∅ 3 µm, Polysciences) are injected in the micro-channel at a measured velocity of 100 µm/s. In fig. 3-a, the four fibered lasers are switched off and the beads flow without being trapped. When the fibered lasers are switched on, the four 20 µm spaced traps are rapidly filled with beads as shown in fig. 3-b. Figure 3-c shows that the two fluorescent beads on the left hand side can easily be detected by turning on the excitation laser. The weak fluorescence of the outer bead is due to the non-uniform illumination of the excitation light. Finally, fig. 3-d shows that the beads are carried away by the flow when the traps are turned off.

Fig. 3. Polystyrene beads (∅ 3-4 µm) trapped in a 150 µm wide micro-channel. (a) No bead is trapped. The black arrow indicates the edge of the channel and the white arrow shows the flow direction. (b) The four optical tweezers are switched on and four beads (shown by white arrows) are trapped. (c) The excitation laser is turned on and the two beads on the left hand side start to fluoresce. (d) The optical tweezers are switched off and the four beads are carried away by the flow.

a bbb

ccc ddd

20 µm

3. SINGLE LASER SOURCE TRAPPING 2D AND 3D OPTICAL TRAPS

Optical interference constitutes a powerful way to build assemblies of multiple traps. As pointed out in several publications, sets of traps can be generated by superimposing coherent light beams either through an interferometric setup or by recombining several beams previously fanned-out by diffractive optical elements.3, 4 The latter scheme obviously includes holographic devices.5 3.1 Optical setup Fig. 4. Optical setup for 3-beam interference: beams a, b, and c, form a pyramid with an equilateral triangular base. Lens L3 focuses all three pseudo-gaussian beams at the summit of the pyramid, where they interfere at their respective waists. The imaging system made of a microscope objective relayed by a projection lens conjugates the waist’s plane with a screen located 4 meters away from it.

Front view

M

MO

Sample

M NPBS

M

Beam b M

Table 13.27°

Top view

PBS L1 M

PBS

Fresnel Rhomb

Laser

M

NPBS

PBS

L3

L2

Projection lens

M Screen

Beam a

Beam b

Beam c

λ/2

λ/2

M

M

M

MO + M

Most common trapping experiments require very high numerical aperture microscope objectives in order to build three-dimensional traps. The high spatial frequency components are needed to generate gradient forces sufficient to counteract the scattering forces that push particles forward. 6,7 In some cases a high magnification microscope objective is needed anyway, either for imaging very small trapped objects or, even more so, to collect fluorescence from such objects. However, the use of high numerical aperture microscope objectives is detrimental to the lateral field of view and to the field’s depth in the imaging of trapped (and non-trapped) particles. In other cases, one doesn’t always have the physical space to mount a microscope objective, even a long working distance one. This is typically the case when cold atoms needed to be optically arranged are already entrapped in a magneto-optical trap. They are then confined at the center of a vacuum chamber, most of the time ruining the possibility to set up high numerical aperture(N.A.) optics. Fortunately, for many applications, optical trapping can be achieved without going to the expenditure of high N.A. optics. One typical example is the generalized phase contrast method proposed and developed by Glückstad and coworkers.8, 9 The remaining part of this paper revisits schemes allowing optical trapping without the need for expensive microscope objectives. Intensity gradients generated by interference of several waves constitute more or less complex templates of traps, depending of the respective position of interfering beams and of their number. Figure 4 depicts a classical interference setup made with three identical beams. In order to control the symmetry and strength of the optical wells , the three beams must have identical pathlengths to ensure that beam sizes and shapes are rigorously similar in the overlapping area. Moreover, equivalent pathlengths provide the best possible configuration for maximizing interference contrast, and therefore generate the strongest possible gradients. The power of the frequency doubled Nd-Vanadate cw laser, delivering up to 5 watts at 532 nm, is first controlled with a beam attenuator made of a Fresnel rhomb followed by two polarizing beam splitters. Lens L3 is positioned such as to focus the beams into the cell where the three beams overlap and interfere. Lenses L1 and L2 form an afocal device that allows for modification of the beam diameter. Beam splitter BS splits the incoming beam in two parts: 1/3 of the energy is reflected and 2/3 are transmitted before being split again (50/50) with a non-polarizing beam splitter cube (NPBS). Beams a and b carry the same linear polarization, A half-waveplate (λ/2) rotates the polarization of beam c to make it consistent with the polarizations of beam a and b. Considering the geometry, the 3 polarization directions cannot be identical; they are tilted by a small slant angle (13.272° in the configuration leading to 2 micron fringes). The 3 beams are folded several times with dielectric mirrors M such as to form a pyramid with a vertical axis. The base of the pyramid is, in most cases but not necessarily, an equilateral triangle. The summit of the pyramid is located at the focal plane of the 3 focused beams: therefore the 3 waists overlap, forming the coherent interference pattern of three plane waves. The energy distribution displays then the shape of a honeycomb material, as shown in figures 5-a and 5-b. Polystyrene spheres of a size matching that of the template created by the 3 beam interference (2 microns in our case) are floating in water in a 50 x 10 x 0.2 mm cell centered around the focal area of the beams. The high refractive index particles are pushed by the scattering force towards the top of the 200 micron thick cell. Meanwhile, they are trapped laterally in the hexagonal structure by the gradient force related to the light intensity

a b c Fig 5. Interference of 3 coherent beams: transverse section of the honeycomb structure

(a) simulation – (b) experimental superimposition of 3 gaussian waists …..(c) 2D optical crystal made of 2 µm polystyrene spheres trapped in b

distribution, The arrangement of many beads in multiple traps can be appreciated by observing the diffracted far field. It is also displayed by an imaging system made of a low numerical aperture microscope (long working distance Nachet, X 20, N.A. = 0.3) relayed by a projection lens. The microscope objective images the whole field, covering more than 1 million optical wells when Lenses L1, L2 and L3 are removed. Only about 1500 optical traps are imaged through the aperture of the projection lens in the actual configuration. The sample is illuminated either by an external laser beam running along the axis of the pyramid, typically at a wavelength different from the one of the trapping laser, or simply by one or several of the trapping beams. Figure 5-c shows a portion of the optical wells filled up with 2 micron polystyrene spheres. The beads scatter forcefuly and obviously interfere with the wells in which they are trapped, thus modifying the phase and the aspect of the image. 3.2 Two-dimensional optical crystals The interference pattern can be either periodic or not. It can easily be made by holographic schemes, as indicated in references 4 and 5. Five coherent plane waves equally distributed on a pyramid with a pentagonal basis lead to an optical Penrose tiling, the 5-fold symmetry being simply imposed by the setup’s geometry. A 3-beam set-up such as the one presented above offers many possibilities: it allows the production of a full set of two -dimensional traps. It can be used with only two beams turned on at a given time, giving the opportunity to change the direction of the interference pattern by switching from one pair of beams to any other pair, thus reconfiguring the beads distribution among various templates of traps. Modifying the size of traps is relatively easy, starting from a given setup, by imaging the existing interference area to a desired magnification. The physics of incommensurate situations can be observed in action by using bead sizes that do not fit the traps’ size. Of course, one can make fixed copies of optical crystals by polymerizing a solution in which beads are trapped. This produces some degree of distortions due to stresses occurring during the gelification process (see e.g. ref 4). 3.3 Three-Dimensional optical crystals The setup described previously takes advantage of the scattering force to push all particles to the top of the cell, helping them to lay on a plane defined by the bottom of the cell’s cover. In order to trap in the third dimension, that is to say along the vertical axis of the setup, it becomes necessary to counteract the scattering force. A simplified concept calls for rigorously balancing the radiation pressure by sending counter propagating beams into the cell: to cancel the scattering force induced by the three incoming beams. One can think of sending back into the cell similar beams with identical divergence and identical waist size. A spherical mirror with the appropriate radius of curvature could produce such retro-beams. However the perturbation created by the presence of trapped spheres modifies the field such that the returned beams would not exactly match the incoming ones. Let’s indicate two other methods allowing the production of arrays of 3D traps. 3.3.1 Talbot trapping

Self images, also called Talbot images, are produced when a periodic structure is illuminated with a plane wave of coherent light. They appear in planes perpendicular to the planar structure, at regularly spaced distances equal to 2d2/λ, with λ being the wavelength of light and d the spatial period of the structure.

Fig.6. (a) SEM of phase Ronchi ruling printed in quartz. The phase shift between adjacent pixels is π. Pixels re 3 µ wide

(b) A few 3 µ polystyrene spheres trapped in Talbot images

a b

In between those plans, at distances equal to (2n + 1)d2/λ lay self images with an inverted contrast. Consequently, a simple illumination of a cross-grating by a collimated laser beam produces a 3D light intensity distribution resembling a three-dimensional mesh. The edge of such a Talbot plate engraved in quartz to create a cross phase Ronchi ruling is represented in figure 6 -a. The part of the figure showing only straight lines shows how the cross grating was manufactured, first by etching a linear phase Ronchi ruling, then rotating the support of 90° and by repeating the engraving process on the direction orthogonal to the first ruling. Fig 6-b show a few spheres trapped in Talbot cages. This self imaging method reconstructs images with a very high spatial frequencies content, which produces steep gradients, corresponding to extremely stiff traps. The attempt we made with a “phase Ronchi plate” manufactured at MIT Lincoln Lab by M. Holtz and co-workers led to extremely strong trapping (see details in reference 4).

3.3.2 Trapping in speckles Speckle pattern is intrinsically three-dimensional and speckle grain sizes obey a well defined Poisson statistic. Speckle intensity always displays high contrast. The size difference between the length and the cross-section of speckle grains has been taken into advantage to trap cold cesium atoms and to get a different cooling efficiency along the long direction rather than along the short one.10

3.3.3 Trapping with diffractive structure s The phase structure used in the above experiments sho ws one original way to utilize diffractive structures. In the following example, a different approach employs a phase structure to diffract 3 beams when being illuminated perpendicularly to its plane. A lens recombines those 3 beams: they interfere to form an hexagonal pattern in a magneto-optical trap holding cold cesium atoms. The set of hexagonal tubes being created with a laser far-detuned from the resonance of the cesium atoms, it allows them to be trapped and distributed in a regular pattern.11,12 Figure 7 displays an image of the atoms trapped in the hexagonal lattice . Many examples of ensembles of 3d traps have been published, using various schemes and numerous types of static and dynamic diffracting structures. One of the most striking applications allows sorting particles by size or by refractive indices.13 Large areas of traps, made simply with amplitude Ronchi rulings cover several square millimeters and perform with relatively moderate laser power, as demonstrated recently.14

3.4 Opti cal binding Any trapped object scatters light. Dielectrics can be pictured as absorbing and immediately re-emitting light. The field radiated by any trapped particles interferes with the field used to perform the trapping, de facto modifying the light intensity distribution in the vincinity of trapped bodies; the original beam-shape utilized to trap is changed as soon as trapping takes place, whatever this original shape is (e.g. gaussian or interferometric pattern assembly). This effect considerably influences the trapping behavior particularly when trapped particles are close to each other: the more objects get trapped, the more they affect the structure they build through their mutual interactions. Since the interaction takes place by exchange of photons, it has been coined “optical binding”. The binding force , first calculated in the simple case of the interaction of two dipoles, one being phase retarded with the other, has been verified

Fig. 7. Absorption image of Cs atoms arranged in a 25 microns hexagonal lattice

experimentally in the Mie regime with two particles located in a one-dimensional trap.15 The calculation made in the dipole case applies to the Rayleigh regime and deals with trapping as a gradient force self-induced by the radiating trapped particles. Another binding situation, first described by Roosen and Imbert, relies on the trap generated by big spheres (in the range of 5 micron diameter) that focus light: other spheres can be trapped in the focal area, and they focus light themselves, then generating another optical well. Short chains of particles have been observed.16,17 Recently, two groups independently revisited one-dimensional binding situation in the Mie regime, where the cohesion force appears to rely more on the scattering force than on the gradient force. 18,19 They created “optical molecules”, forming chains of optically bound matter. Results show very clearly self assembly configurations being dependent of the size , number, and optical indices of the undertested particles. It should be pointed out that it becomes difficult to separate the origin of forces into gradient force and scattering force in any situation involving the Mie regime. As an example, figure 8 shows a facetted 2D optical crystal created simply by throwing 3 micron polystyrene spheres in a wide pseudo-gaussian well (about x microns in diameter) in a setup similar to the one described earlier but involving only one laser beam. All spheres are pushed by the scattering force to the top of the cell, and they all should seek a minimum energy configuration by trying to go to the center part of the beam. Any sphere coming near an already formed crystal “sees” the wells generated by the interference pattern due to the many body interaction. It hops from well to well until reaching the close vincinity of the crystal, where it interacts with the whole ensemble that gets re-organized until it reaches a minimum energy situation. This example dramatically shows the importance of optical binding when many objects are trapped in wells separated by distances in the range of one to a few wavelengths. We have seen many experimental evidences showing that the binding force can become preponderant over the trapping force in cases where one expects to arrange many spheres according to a trapping template. A re-organization of the sphere distribution takes place, leading to unexpected structures such as super-gratings. All examples of optical binding described in this paper correspond to one or two -dimentional structures. The effect is obviously of a three-dimensional nature.

4. CONCLUSIONS Sets of multiple optical traps differ considerably depending on their configuration and on the methods utilized to produce them. The use of relatively widely spaced traps made with separate lasers leads to total flexibility and to strong optical independence from the undertested specimen. Conversely, interferometric setups present a packaging aspect allowing better space filling, but generate arrays of traps in which many samples can interact through photon exchanges. This effect can be detrimental in some cases, but can also be taken advantage of to strenghten trapping stability, and to generate self assembling arrangements. Diffractive optical elements can be built with various designs producing many different patterns and can be used along varoius schemes to produce independent sets of traps as well as correlated ones,

Fig.8. Self assembled optical crystal illuminated by a single laser beam forming a large (30 µm) gaussian trap. The multiple coherent scattering of all (3 µm) spheres generates sets of fringes through its interference with the orig inal gaussian trap. Any bead approaching this crystal will be trapped in those fringes and will contribute to modify the total intensity distribution

scanning a whole pal let of possibilities. Existing optical trapping schemes can produce random trap sizes and trap positions as well as periodic and quasi-periodic arrays. The diversity of shapes, the endless possibilities for trap distribution, and the number of controling parameters such as intensity, polarization, size, wavelength, etc, opens the door to a world of applications in many fields.

5. REFERENCES

1. D. C. Duffy, J. C McDonald, 0. J. A. Schueller and G. M. Whitesides, “Rapid Prototyping of Microfluidic Systems in Polydimethylsiloxane”, Anal. Chem., 70, pp. 4974-4984, 1998.

2. K. Svoboda, S. M. Block, “Biological applications of optical forces”, Ann. Rev. Biophys. Biomol. Struct., 23, pp. 247-285, 1994.

3. M.M. Burns, J.-M. Fournier, and J.A. Golovchenko, “Optical Matter, Crystallization and Binding in Intense Optical Fields”, Science, 249, pp. 749-754, 1990.

4. J.-M. Fournier, M.M. Burns, and J.A. Golovchenko, “Writing Diffractive Structures by Optical Trapping”, Proc. SPIE 2406, pp. 101-111, 1995.

5. M.M. Burns, J.-M. Fournier, and J.A. Golovchenko, "Optical Matter" , US Patent # 5,245,466, 1993.

6. A. Ashkin , “Acceleration and Trapping of Particles by Radiation Pressure”, Phys. Rev. Lett., 24, n° 4, pp, 156-159, 1970.

7. A. Ashkin, “Forces of a single beam gradient laser trap on a dielectric sphere in a ray optics regime”, J. of Biophys. Soc., 61, pp. 569-582, 1992.

8. R.L. Eriksen, P.C Mogensen,.J. Glückstad, “Multiple-beam optical tweezers generated by the generalized phase-contrast method”, Opt. Lett., 27, pp. 267-269, 2002.

9. V.R. Daria, R.L. Eriksen, S. Sinzinger, J. Glückstad, “Optimizing the generalized phase contrast method in a plan ar-optical device”, Journ of Optics A: Pure and Appl Opt , 5, pp. s211-s215, 2003.

10. D. Boiron, C. Mennerat-Robillard, J.-M. Fournier, L. Guidoni, C. Salomon, and G. Grynberg, “Trapping and cooling cesium atoms in a speckle field”, Eur. Phys. J., D 7, 373-377, 1999.

11. D. Boiron, A. Michaud, J-M. Fournier, L. Simard, M. Sprenger, G. Grynberg, and C. Salomon, "Cold and dense cesium clouds in far-detuned dipole traps" , Phys. Rev. A, 57, R4106-R4109, 1998.

D. Boiron, A. Michaud, J.-M. Fournier, L. Simard, G. Grynberg, and C. Salomon, "Strong confinement of cesium atoms in far-detuned dipole traps, lattices, and speckle patterns" , Proc. of CLEO/Europe-EQEC’98, 1998.

12. C. Mennerat-Robilliard, D. Boiron, J.-M. Fournier, A. Aradian, P. Horak, and G. Grynberg, "Cooling cesium atoms in a Talbot lattice", Europhysics Letters, 44, 442-448, 1998.

13. M. P. MacDonald, G. C. Spalding, K. Dholakia, "Microfluidic Sorting in an Optical Lattice”, Nature 426, pp.421-424, 2003.

14- G.C. Spalding, V. Garcés-Chávez, K. Dholakia, “Guiding and trapping microparticles in an extended surface field”, SPIE 5514, 2004.

15. M.M. Burns, J.-M. Fournier, and J.A. Golovchenko, "Optical Binding", Phys. Rev. Lett., 63, pp. 1233-1236, 1989.

16. G. Roosen, "La lévitation optique de sphères”, Can. J. Phys. 57, pp.1260-1279, 1979 ; G Roosen, B. Delaunay, and C. Imbert, “Radiation pressure exerted by a light beam on refractive spheres: theoretical and

experimental study”, J. Opt. 8, pp.181-187, 1977.

17. G. Roosen and C. Imbert, “Optical levitation by means of two horizontal laser beams: a theoretical and experimental study”, Phys.Lett A, 59A, pp. 6-8, 1976;

G. Roosen and S. Slansky, “Influence of the beam divergence on the exerted force of a sphere by a laser beam and required conditions for stable optical levitation”, Opt. Comm. 29, pp. 341-346, 1979.

18. S.A: Tartarkova, A.E: Carruthers and K. Dholakia, “One dimensional optically bound arrays of microscopic particles”, Phys. Rev. Lett, 89, pp. 283901, 2002.

19. W. Singer, M. Frick, S. Bernet and M. Ritsch-Marte, “Self-organized array of regularly spaced microbeads in a fiber-optical trap”, J. Opt. Soc. Am. B 20, pp. 1568-1574, 2003.