Design and Characterization of Integrated Optical Devices for Biophotonics

Transcription

1 UNIVERSITA DEGLI STUDI DI PAVIA FACOLTA DI INGEGNERIA Dottorato di Ricerca in Ingegneria Elettronica, Informatica ed Elettrica XXIII Ciclo Design and Characterization of Integrated Optical Devices for Biophotonics Tesi di Dottorato di Lorenzo Ferrara Anno 2010

2 Contents INTRODUCTION... 4 CHAPTER 1 INTRODUCTION ON SINGLE CELL MANIPULATION THROUGH OPTICAL FORCES ORIGIN OF OPTICAL FORCES RAYLEIGH REGIME (a << λ/20) LARGE PARTICLES REGIME (a >> λ) OPTICAL TRAP SINGLE-BEAM OPTICAL TRAP DUAL-BEAM OPTICAL TRAP CHAPTER 2 OPTICAL TWEEZERS OPTICAL TWEEZERS IN LITERATURE STANDARD OPTICAL TWEEZERS FIBER OPTIC TWEEZERS TOTAL INTERNAL REFLECTION FIBER OPTICAL TWEEZERS WORKING PRINCIPLE NUMERICAL ANALYSIS NUMERICAL RESULTS: TRAPPING FORCES NUMERICAL RESULTS: ESCAPE ENERGY FABRICATION OF A TOTAL INTERNAL REFLECTION OPTICAL FIBER TWEEZERS FABRICATION OF THE FOUR-FIBER BUNDLE FABRICATION BY FOCUSED ION BEAM FABRICATION BY TWO PHOTON LITOGRAPHY EXPERIMENTAL RESULTS FIB-FABRICATED TOFT: EXPERIMENTAL RESULTS TWO-PHOTON LITHOGRAPHY FABRICATED TOFT: EXPERIMENTAL RESULTS CHAPTER 3 DISCRETE ELEMENTS OPTICAL STRETCHER INTRODUCTION CELL MECHANICAL PROPERTIES EXPERIMENTAL TECHNIQUES FOR PROBING CELL MECHANICAL PROPERTIES EXPERIMENTAL SETUP OPTICAL PART FLUIDIC PART EXPERIMENTAL RESULTS RESULTS ANALYSIS... 66

3 3.3.2 EXPERIMENTAL RESULTS ON RED BLOOD CELLS EXPERIMENTAL RESULTS ON CANCER CELLS CHAPTER 4 INTEGRATED OPTICAL STRETCHER STRUCTURE OF AN INTEGRATED OPTICAL STRETCHER DESIGN SIMULATIONS FABRICATION EXPERIMENTS EXPERIMENTAL RESULTS WITH A ROUND-SECTION MICROCHANNEL FABRICATION OF A SQUARE-SECTION MICROCHANNEL AND EXPERIMENTAL RESULTS CONCLUSIONS APPENDIX A MATLAB PROGRAMS BIBLIOGRAPHY

4 INTRODUCTION In molecular and cellular biology an impelling demand has arisen for the development of tools able to select, isolate and monitor single cells or cell clusters. Experiments on single cells have the potential to uncover information that would not be possible to obtain with traditional biological techniques, which only reflect the average behavior of a population of cells. In the averaging process, information regarding heterogeneity and cellular dynamics, that may give rise to a nondeterministic behavior at the population level, is lost. Obvious reasons for the existence of this heterogeneity are different genotypes and variations due to the cell cycle stage or age; even in a monoclonal population, with the same history and in the same environment, different phenotypes can exist due to the Stochastic nature of gene expression. Thus it is necessary to perform experiments on a single cell level, in order to determine how the cells really react and thus get a complete picture of how cells function. The exploitation of optical forces represents an accurate, non-invasive and gentle manipulation technique for individual cell studies. Nowadays there are two biophotonic tools that can provide complementary information on cell properties: the optical tweezers (OT) and the optical stretcher (OS). OT allow easy trapping and manipulation of individual cells using a laser beam heavily focused and, combined with fluorescence analysis, represents a flexible tool for cell monitoring and sorting. OS relies on a double-beam trap obtained through two counterpropagating fiber beams. The radiation pressure exerted by the two beams is perfectly counterbalanced so that the total force acting on the centre of mass of a trapped cell is zero. However the stress distributed over the cell surface can cause deformation on the cell. By increasing the laser power, the cell elongation along the beam axis becomes measurable. It is a powerful device for the investigation of cell mechanical properties that can open new scenarios for the comprehension of the basic biological mechanisms and for the early detection of several diseases. The goal of my thesis work is the fabrication and improvement of such novel devices, supported by a dedicated numerical model, based on a ray-optics approach, able to provide an accurate description of the optical forces and to identify the optimal fabrication parameters. The exploitation of optical tweezers in all-fiber technology for trapping and manipulation of biological specimens would represent a real break-through in many applications, overcoming most of the problems related to the bulky structure of standard-tweezers based on an optical microscope; the development of a fully integrated optical stretcher (FIOS), which is the integration on the same

5 chip of both microfluidic and optical functions for optical stretching could lead to a simple and miniaturized optical device suitable for real medical analysis. My thesis is structured as follows. In Chapter 1 I explain the origin of optical forces and their interaction with a spherical object in Rayleigh and Large Particle regime. Then I show how to obtain an optical trap, with a single focused beam or with two counterpropagating beams. In Chapter 2 the standard optical tweezers and the first developments of fiber optical tweezers are introduced, describing their structures, advantages and limitations. Then I show the working principle of the fiber tweezers proposed in our laboratory: the TOFT device (Total internal reflection optical fiber tweezers). It is the first single-fiber tweezers able to guarantee 3D trapping with a long working distance based on a new approach that combines two concepts: i) exploitation of fiber bundles, ii) achievement of tight focusing by total-internal-reflection at the microstructured fiber end-faces. Two strategies for the end-fiber microstructuration are investigated: micromachining through focused ion beam and two photon polymerization lithography. For each fabrication I will report the experiments and results achieved. Chapter 3 describes the importance of cell mechanical properties in the biological field and the methods to measure them. I introduce the theory of the optical stretcher and its implementation with discrete elements. This device has been used in the frame of different collaborations with biologists and medics; I ll describe either the results obtained with the analysis of red blood cells of diabetic and anaemic patients, or the experiments performed on metastatic lymphocytes and fibroblasts. Finally, Chapter 4 describes the design and fabrication of the fully integrated optical stretcher, accomplished by fabricating waveguides and microfluidic channels on the same substrate of fused silica through a recently developed technique based on femtosecond laser writing. This FLICE (Femtosecond Laser Irradiation followed by Chemical Etching) technique is very simple and practical and, combined with chemical etching, guarantees extreme flexibility and 3D capabilities. A first implementation of FIOS with a round section of the microchannel is tested probing the viscoelastic properties of red blood cells. A second implementation of FIOS, with a square section microchannel, is also described, as well as the characterization of the trapping force and the results in red blood cells stretching.

6 CHAPTER 1 INTRODUCTION ON SINGLE CELL MANIPULATION THROUGH OPTICAL FORCES This chapter briefly reviews the physical principles at the basis of the mechanism for single cell manipulation without physical contact. The origin of optical forces will be first described and secondly different configuration of optical traps exploiting optical forces will be analyzed. 1.1 ORIGIN OF OPTICAL FORCES Since the beginning of the seventeenth century, the German astronomer Johannes Kepler proposed that the reason why comet tails point away from the sun is because they are pushed in that direction by the sun s radiation. In 1873, James Clerk Maxwell predicted in his theory of electromagnetism that light itself can exert an optical force, or radiation pressure, when hitting an object. Anyway this effect was not demonstrated experimentally until the turn of the century since radiation pressure is extraordinarily feeble; indeed milliwatts of power impinging on an object produce forces that are only in the order of piconewtons. The advent of lasers in the 1960s finally enabled researchers to study radiation pressure through the use of intense, collimated sources of light. The pioneer of such studies was Arthur Ashkin who, with his coworkers, demonstrated that, by focusing laser light down into narrow beams, small particles, such as few micron-diameter polystyrene spheres, could be trapped, displaced and even levitated against gravity using the force of radiation pressure [1] 1. The effect of optical forces on macroscopic objects can be disregarded, since their weight is much higher than the intensity of optical forces. Anyway these forces become significant on the scale of macromolecules, organelles, and even whole cells, whose mass is in the order of kg. A force of ten piconewtons can indeed tow a bacterium through water faster than it can swim, halt a swimming sperm cell in its track, or arrest the transport of an intracellular vesicle. A force of this magnitude can also stretch, bend, or otherwise distort single macromolecules, such as DNA and RNA, or macromolecular assemblies, including cytoskeletal components such as microtubules and actin filaments. 1 A. Ashkin, Acceleration and trapping of particles by radiation pressure, Physical Review Letters, Vol. 24, No. 4, 1970

7 The origin of the optical forces exerted on a dielectric particle can be ascribed to the momentum transfer resulting from the refraction and reflection of the incident photons. The interaction between the electromagnetic radiation and a particle it is based on the radiation scattered by the particle itself. To better describe this interaction, it s worth to consider two different scattering regimes on the basis of the ratio between the particle dimension and the radiation wavelength RAYLEIGH REGIME (a << λ/20) When the particle dimension is smaller than the wavelength, the optical forces can be calculated following the Rayleigh approximation. Under this condition the particle is treated as an induced small dipole immersed in an optical field oscillating at frequency ν. The forces acting on this dipole are of two species: (i) the scattering (or radiative) force originated by momentum changes of the light caused by scattering, and (ii) the gradient (or dipole) force due to the Lorentz force acting on the induced dipole. The scattering force is proportional to the laser intensity and its effect is to push the particle along the laser beam propagation (z-axis in our consideration) while the gradient force moves the particle toward the gradient of the optical intensity. Figure 1.1: Sketch of a particle in the Rayleigh regime Since a dipole has a proper resonance frequency ν 0 the induced dipole is attracted toward the region of maximum intensity or it is repelled from it according to the forcing optical field, in particular if it is red-detuned (ν < ν 0 ) or blue-detuned (ν > ν 0 ). Within the zeroth-order approximation in a paraxial Gaussian beam description (λ << ω 0 ) the scattering and gradient force produced by a laser beam are given by Florin et al. [2] 2 F scatt r n2 zˆ c a m m I r (1) 2 E.L. Florin, A. Pralle, E.H.K. Stelzer, J.K.H. Horber, Appl. Phys. A 66 (1998) S75.

8 2 2 3 m 1 2 F grad r 2 n2 0a E r 2 m 2 (2) where the position vector r is referred to the beam center at the minimum waist, ž is the unit vector along the z-axis, ε 0 the dielectric constant in the vacuum, c is the speed of light, and m is the ratio between the refractive index of the particle n 2, and that of the surrounding medium n 1 (m = n 2 /n 1 ). The intensity I(r) is defined as a time-average of the Poynting vector S(r, t) which is related to the electric and magnetic field components by I r zˆ S r, t Re E r H r * n2 0c 2 E r zˆ 1 (3) T 2 2 In presence of strongly focused laser beams, higher-order contributions are required for describing the transverse and longitudinal components of the electric and magnetic fields LARGE PARTICLES REGIME (a >> λ) In the large particles regime, commonly referred to as Mie regime, the particle size is much larger than the radiation wavelength (a >> ). In this case, since the particle is not affected by temporal variations of the electric field, we can describe the interaction between radiation and particle through a ray optics approach. The electromagnetic beam is then decomposed in a set of rays, each one carrying a fraction of the total power of the optical beam. The behavior of optical rays, when crossing the interface between two media having different refractive index (n1 and n2), can be evaluated by Snell's law to calculate the propagation direction 1 sin 1 n2 sin n (4) 2 and by Fresnel relations to calculate, depending on the polarization of the radiation, the amount of power that is reflected from the surface.

9 r n cos 2 n2 cos 1 n cos 1 (5) n1 cos r n cos 1 n cos (6) n1 cos 1 n 2 cos 2 In this way the beam can be described by geometrical considerations on the direction of the rays. Figure 1.2: Sketch of a particle immersed in an electric field in the case of large particle regime. In order to describe the effect of radiation pressure, we consider a spherical particle with refractive index n 2 immersed in a homogeneous medium of index n 1, and we analyze only the effect of a single ray composing the Gaussian beam. We also suppose that the radiation wavelength is such that the optical absorption of the particle is negligible. When a ray hits the interface between two different media it gives rise to two components, one reflected and one transmitted, each one carrying a small portion dw of the total power of the beam, as shown in Figure 3. Indicating with the angle between the ray incident on the particle and the normal to the surface, and with n the refractive index of the particle normalized to the external medium n = n2/n1, the angle of refraction is given by Snell's law: sin sin (7) n To evaluate the power associated to the transmitted and the reflected rays is necessary to use the Fresnel coefficients (Eq. 1.2 and 1.3). We will indicate with R and T respectively the reflection and

10 the transmission coefficients of the beam power, which are related to the Fresnel coefficients by the relations R = ρ ς, π 2 e T = 1-R. Figure 1.3: Ray optics description of the behaviour of an optical ray hitting a spherical particle. The light beam transmitted into the first interface hits the inner surface of the sphere and divides into two components, one reflected back into the particle and one transmitted outside. This process creates an infinite number of rays gradually decreasing in optical power that emerges from the sphere at different points and with different propagation directions. In particular, being dw the power of the incident ray, it follows that the first reflected ray will have a power of RdW, while the rays transmitted after passing through the particle will have a gradually decreasing power of T 2 dw, RT 2 dw, R 2 T 2 dw, etc. It is possible to describe the forces that are exerted at each point of refraction, and decompose them into the components along z and y axis. It should be noted that the first beam is reflected at an angle equal to π+2θ with the z axis, while the successive rays transmitted form an angle respectively equal to α, α+β. Α+2β,... with the direction of incidence, where the values of and are retrievable through purely geometrical considerations: 2 (8) 2 (9) Adding the terms of the force along z and y axis produced by the individual rays we obtain: n1dw n1dw n1dw 2 2 F z Rcos 2 T cos Rcos R cos 2... (10) c c c

11 n1dw n1dw 2 2 F y Rsin 2 T sin Rsin R sin 2... (11) c c Using a complex notation F TOT = Fz + ify and an exponential notation we obtain: F TOT n dw 1 R c 2 j i j cos 2 isin T R e 1 2 j 0 (12) This series highlights that the contribution of force related to the transmitted beam decreases as a geometric series, hence we can obtain the following expression: F TOT i n 1dW 2 1 i e e R c 1 e R T i 2 (13) Inserting the values of and described above and dividing the force in real and imaginary components (respectively along z and y directions), it is possible to retrieve the final formula of the force produced by a beam having optical power dw 2 R cos 2 R 2R cos 2 2 n 1dW T cos 2 F S 1 R cos 2 2 (14) c 1 2 R sin 2 2R cos 2 2 n 1dW T sin cos 2 F G R sin 2 2 (15) c 1 R These equations give a quantitative estimate of the forces, and highlight the generation by a light beam of two distinct components of the forces, one parallel and the other perpendicular to the beam direction, which, by following the description given for the Rayleigh regime, can be respectively called "scattering force" and "gradient force. The effect of the first component is to push the particle along the beam direction, while the second tends to pull the particle towards the center of the beam, where the intensity is higher. 1.2 OPTICAL TRAP

12 An optical trap is a stable equilibrium point in space of the optical forces acting on a particle. If we consider a dielectric particle near the axis of a laser beam, it will experience a force because of the transfer of momentum from the scattering of photons incident on the particle to the particle itself. The resulting optical force, as described above, can be decomposed into two components: a scattering force, parallel to the light propagation, and a gradient force, in the direction of the spatial light gradient, i.e. perpendicular to the propagation direction SINGLE-BEAM OPTICAL TRAP For most conventional situations, the scattering force dominates. However, if there is a steep intensity gradient, as it happens near the focus of a tightly focused laser beam, the second component of the optical force, the gradient force, is no more negligible and must be taken into account. When the axial gradient component of the force, which pulls the particle towards the focal region, is equal to the scattering component of the force, which pushes it away, a stable trapping in all three dimensions is achieved. In order to fulfill this condition a very steep gradient in the light intensity is needed and it might be produced by sharply focusing the trapping laser beam to a diffraction-limited spot by means of a high NA objective. As a result of this balance between the gradient and the scattering force, the axial equilibrium position of a trapped particle is located slightly beyond the focal point. For small displacements from the equilibrium position, the gradient restoring force is simply proportional to the offset from the equilibrium position, i.e., the optical trap acts as Hookean spring whose characteristic stiffness is proportional to the light intensity.

13 Figure 1.4: Schematic of a single-beam laser trap. The variation of photons momentum originates a force on the particle that pulls it towards the focus of the beam. Refraction of the incident light by the sphere corresponds to a change in the momentum carried by the light, hence to a force F = dp / dt. By Newton s third law, an equal and opposite force proportional to the light intensity is imparted to the sphere. When the particle s refractive index is greater than that of the surrounding medium, the optical force arising from refraction is directed as the intensity gradient. Conversely, for an index lower than that of the medium, the force is in the opposite direction with respect to the intensity gradient. In the case of a uniform sphere in large particle regime, optical forces can be directly calculated in the ray-optics regime by decomposing the laser beams into optical rays. As shown in Figure 1.4, the rays that hit the particle change their propagation direction because of refraction at the interfaces. The external rays contribute to the axial gradient force, whereas the central rays are primarily responsible for the scattering force. Thanks to the strong focusing of high NA objectives the gradient force can be sufficiently strong to counterbalance the scattering force, anyway an expansion of the Gaussian laser beam, to slightly overfill the objective entrance pupil, can increase the ratio of gradient to scattering force, resulting in improved trapping efficiency. Depending on the position of the particle center O respect to the beam focus, the resulting trapping force will pull the particle in different directions so as to move the particle towards the equilibrium position, as schematically shown in Figure 1.5.

14 Figure 1.5: Schematic of the optical trapping force direction acting when the particle is displaced from the equilibrium trap position [3] DUAL-BEAM OPTICAL TRAP As proposed by A. Ashkin [1], a stable optical trap can also be achieved by exploiting two equal counterpropagating Gaussian beams, see Figure 1.6. Figure 1.6: Schematic of a dual-beam laser trap, as proposed by A. Ashkin [1]. In this case the gradient forces pull the particle towards the axis of the beams while the scattering forces push the particle in the middle of the two beams where they re balanced. In this condition, the trapping setup is similar to a spring system. In fact we can assume the case in which the particle is translated in the z direction, along the beam axis. if P is the power transmitted by the fibers, and the light from the fibers can be represented by a Gaussian beam with waist w 0 at the fibers face, then the intensity at the fiber faces is given by l 0 = 2P/(πw 2 0 ). For a plane wave with intensity I, the scattering force on a sphere of radius R is given by (rπ 2 )IQ pr /c. Let z = 0 at the center point between the fibers. Using an approximation in which the intensity and the phase of the trapping beams are constant over the area of a sphere with radius R, we can express the total scattering force as 3 A. Ashkin, Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime, Biophysical Journal, Vol. 61, 1992

15 F s ap Q r pr, r w 2 0r ap Q pr, g 2 0g 2 S 2 z 1 d 2 S d r g z g w (16) where a = 2R 2 /c, d -1 = λ/(πw 2 o ), S is the separation between the two fiber faces, and A and Q are the wavelength and radiation pressure coefficient, respectively, for the designated color. Let z eq be the value of z for which the force given in the above equation is zero. When a particle is displaced from Z eq, a restoring force results from the increase in intensity with decreasing distance from the fiber face. This restoring force is simply a manifestation of the scattering force and can be expanded to first order in ε = z - z eq, resulting an equation of the form F = -ke, where k is given by k 2 2 P 2 gq pr, g w0g Pr Q pr, r w0r 16 as (17) g S 4d g r S 4d r 2 k as a function of S is a maximum when S is approximately twice the Rayleigh range. The above discussion assumes that the two trapping beams are exactly counter-propagating, i.e. that the two optical fibers are perfectly aligned. However, as shown in Figure 1.7, there are two possible types of fiber misalignment: a positional misalignment, in which the beams are propagating in the ±z direction but the two fibers are translationally displaced, and a rotational misalignment, where both fiber faces still have their centers on the z axis but are at skewed angles to each others' faces and therefore the two light beams are not counter-propagating. Both types of misalignment may occur at the same time.

16 Figure 1.7: (a) Schematic of the forces for each of the two fibers that compose the trap. (b) (d) Directions of the total forces when the fibers are (b) perfectly aligned, (c) translationally misaligned, and (d) rotationally misaligned [4] 4. The alignment of the two counter-propagating beams to within a fraction of the beam waist is the critical point for good trapping operation. For example, if the fibers are translationally misaligned, then the particle can oscillate back and forth between the two fiber faces instead of finding a stable trap position. It is worth to note that even a misalignment translation of 2 m between the two fibers causes a significant decrease in the trapping efficiency. 4 A. Constable et al., Demonstration of a fiber-optical light-force trap, Optics Letters, Vol. 18, No. 21, 1993

17 CHAPTER 2 OPTICAL TWEEZERS This chapter reviews some of the different configurations of the single-beam optical tweezers. After a brief introduction on the tweezers presented in literature, our proposal to realize a fiber optic tweezers is introduced. Working principle, fabrication techniques and experimental test are presented. 2.1 OPTICAL TWEEZERS IN LITERATURE Optical tweezers allows trapping and manipulating singe cells without physical contact. Because of this peculiar feature they are widely used for many different applications, above all in the biological field. For this reason it is interesting to review some implementations of optical tweezer starting from the first proposal STANDARD OPTICAL TWEEZERS Nowadays many implementations of an optical tweezers are available and there is still a great effort in researching new improved solutions. The simplest configuration uses a Gaussian laser beam, expanded by a system of thin lenses. The beam is then reflected by a mirror and focused on the sample by a microscope objective [Fig. 2.1]. Fig. 2.1: Basic principle of single-beam optical tweezers

18 The first working optical trapping scheme, proposed in 1978 and demonstrated in 1986 by A. Ashkin [5 5, 6 6 ], simply consisted in bringing a laser beam to a diffraction-limited focus using a good lens, such as a microscope objective. Figure 2.2 shows the schematic of this optical tweezers. Fig. 2.2: Schematics of a standard Optical Tweezers The alignement of the beam with the objective might be critical, hence the beam exiting the laser usually requires to be expanded in order to overfill the back aperture of the objective; for a Gaussian beam, the beam waist is chosen to roughly match the objective back aperture. A simple Keplerian telescope is sufficient to expand the beam, then a second telescope, typically in a 1:1 configuration, is used for manually steering the position of the optical trap in the specimen plane. If the telescope is built such that the second lens, L4 in Figure 2.1, images the first lens, L3, onto the back aperture of the objective, then a movement of L3 lens corresponds to a movement of the 5 Ashkin A Trapping of atoms by resonance radiation pressure. Phys.Rev. Lett. 40: Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S Observation of a singlebeam gradient force optical trap for dielectric particles. Opt. Lett. 11:288-90

19 optical trap in the specimen plane with minimal perturbation of the beam. Because lens L3 is optically conjugate to the back aperture of the objective, motion of L3 rotates the beam at the aperture, which results in translation in the specimen plane with minimal beam clipping. If lens L3 is not conjugate to the back aperture, then translating it leads to a combination of rotation and translation at the aperture, thereby clipping the beam. Additionally, changing the spacing between L3 and L4 changes the divergence of the light that enters the objective, and the axial location of the laser focus. Thus, L3 provides manual three-dimensional control over the trap position. The laser light is coupled into the objective by means of a dichroic mirror (DM1), which reflects the laser wavelength, while transmitting the illumination wavelength. The laser beam is brought to a focus by the objective, forming the optical trap. For back focal plane position detection, the position detector is placed in a conjugate plane of the condenser back aperture (condenser iris plane). Forward scattered light is collected by the condenser and coupled onto the position detector by a second dichroic mirror (DM2). Trapped objects are imaged with the objective onto a camera. Dynamic control over the trap position is achieved by placing beam-steering optics in a conjugate plane to the objective back aperture, analogous to the placement of the trap steering lens. For the case of beam-steering optics, the point about which the beam is rotated should be imaged onto the back aperture of the objective. This is the basic setup for an optical tweezers; nowadays it is implemented with additional optics like an acousto-optic deflector that provides time-sharing multi-trap, or a liquid crystals space light modulator (SLM) that allows holographic multitraps in the same time, or with an axicon that transform the Gaussian beam into a Bessel beam, useful to have many trapping point along the beam axis. The particle tracking can be well analyzed by a four-quadrant photodiode instead of a CCD: this technique improve the measurement precision and velocity. Despite optical tweezers have been successfully used in many applications, the bulky structure of standard optical tweezers, as well as the expensive setup, limit their diffusion among biological labs. In addition, the use of standard optical tweezers in turbid media or in thick samples presents significant challenges, being difficult to achieve the tight focusing necessary for optical trapping. The realization of an optical tweezers based on a single optical fiber would turn this device into a miniaturized and handy diagnostic tool, suitable for many relevant applications, like in vivo biological operations, where standard tweezers cannot be successfully exploited.

20 2.1.2 FIBER OPTIC TWEEZERS The realization of optical tweezers based on optical fibers would allow a miniaturized, versatile and handy tool to be obtained, suitable for many applications relevant to biology and fundamental physics, such as in vivo biological manipulation or in-vacuum single-particle X-ray spectroscopy. The typical approach for the development of fiber-optical tweezers makes use of two fibers, as discussed in Chapter 1, aligned so that the laser beams exiting from the fibers are counterpropagating along a common optical axis. In this case, the axial scattering forces are counterbalanced, so that is quite easy to obtain a stable optical trap, but the set-up requires a critical alignment between the two fibers and manipulation in three dimensions is quite limited. A single-fiber approach would solve these problems, but as in the standard optical tweezers configuration, a strong focusing of the laser beam is needed to realize the optical trap. The simplest idea is that of building a lens on top of the fiber. Conventional silica fiber tips can be shaped into tapered lenses, but their performance when immersed in a medium such as water depends critically on the radius of curvature of the fiber tip. Since many important systems are dispersed in water, it would be desirable to have fiber tweezers that could work robustly in such a liquid. Since the refractive index of water n w =1.33 is close to that of silica n s =1.45 the focusing of light is not effective, typically resulting in large spot sizes and small working focal lengths. This renders the optical trap ineffective in applications that require trapping of micron-scale particles. Decreasing the radius of curvature of the fiber tip enhances the focusing of light, but also causes light leakage through the fiber cladding, resulting in a decrease in efficiency of the optical trap. In his paper, Taguchi 7 proposed a fiber tweezers based on a single microlensed optical fiber. Microsphere could be manipulated to the forward and backward, or right and left directions synchronized to the optical fiber. Anyway, being the numerical aperture achieved with this lens insufficient to obtain a real three dimensional trapping, such a solution allowed only two dimensional trapping, being the trapping in the third dimension given by electrostatic phenomena. 7 Taguchi Single laser beam optical trap

21 Fig. 2.3: Side view image of the typical relation between the laser beam axis and optically trapped sphere in the solution proposed by Taguchi [7] On the other hand, Liu 8 obtained a purely optical 3D trapping by means of single fiber through highly tapered fibers. The probe was made from a single mode optical fiber with a core diameter of 9 μm, which was tapered by heating and drawing technology, heating the waist zone of the tapered fiber and drawing at high speed of 1.6 mm/s until the fiber break at the waist point. The parabola-like profile fiber tip was obtained from the surface tension of the fused quartz material. In this case the trapping point gets very close to the fiber tip, making it difficult to trap a particle of large size without physical contact. 8 Liu Tapered fiber optical tweezers for microscopic particle trapping

22 Fig. 2.4: (a) The intensity of the optical field emerging from the fiber probe; (b)yeast cell trapped by a single tapered fiber optical tweezers [8]. 2.2 TOTAL INTERNAL REFLECTION FIBER OPTICAL TWEEZERS In order to obtain an efficient fiber optical tweezers, we proposed a different approach that exploits the total internal reflection phenomenon to achieve the high NA necessary to obtain the optical trap WORKING PRINCIPLE As discussed in chapter 1, to increase the gradient force component, a Gaussian beam must be focused on the particle by a high NA objective so as to counterbalance the scattering force that pushes the particle away. Moreover, considering a strongly focused Gaussian beam in an optical ray regime, the central on-axis rays contribute mainly to the scattering force, yielding a negligible contribution to the axial gradient force. As to suppress the on-axis scattering force, we decided to use a bundle of optical fibers that behaves approximately as a fiber with annular core and we cut the cores of the fibers at an angle θ so that the propagating light experiences total internal reflection (TIR) at the interface with the surrounding medium, as shown in Figure 2.5. Hence optical beams are first deflected into the cladding and then transmitted out of the fibers converging all in the same point, at a large angle with respect to the fiber axis. The resulting structure provides, for optical trapping purposes, the equivalent effect of a focused beam, with the advantage that the scattering force in the axial direction is highly suppressed. We indicate such a tweezer as TIR-based optical fiber tweezer (TOFT).

23 Fig. 2.5: Scheme of the total internal reflection optical fiber tweezers. (a) Cross section of the annular core fiber: the optical beam experiences reflection in correspondence of the fiber cut and refraction at the fiber medium interface. The φ angle determines the equivalent NA of the fiber probe. (b) Annular core fiber: the core is represented with the dark gray area. (c) Optical fiber bundle: the tweezers working principle can also be applied to a fiber bundle provided that the fiber cores (dark gray circles) are symmetrically positioned around the bundle axis. By simple trigonometric considerations, and by using Snell law, it is possible to express the angle of convergence φ through the following relation (n F and n M being the refractive indexes of the fiber and the surrounding medium, respectively): (18) As a consequence, the structure provides a focusing effect corresponding to that obtained using an objective with an equivalent NA given by NA eq = n M sin(φ). The NA eq can be more conveniently expressed as a function of the fiber parameters: (19) Taking n F = 1.45 and considering n M = 1.33 as the refractive index of the surrounding medium (water), by cutting the fiber surfaces at an angle θ slightly beyond the critical angle for TIR (θc = 66.5 ), the structure behaves like an optical system with NAeq = 1.06, a value very close to that of

24 the typical objectives used in bulk optical trapping arrangements. The position of the trapping point can also be easily evaluated through ray optics considerations. If the diameter of the core annulus is, as an example, D = 110 μm, the trapping position is about 40 μm away from the point of TIR, thus allowing a high degree of freedom in sample manipulation. It is possible to fabricate TOFT with bigger bundles; the adding of more fibers increases the final dimension of the probe, but it can exploit additional features. In Fig. 2.6 we show some example of the possible functions that could be realized using the proposed structure. In a) the possibility to realize multiple traps along the probe axis, just by using a different cutting angle θ on three of the fibers, is shown. A possible steering of the fiber beams necessary to realize multiple traps at the same distance from the probe end is shown in b). As depicted in c), it is also interesting to notice that the radiation pressure that can be exerted, on a trapped particle, by using the light output from the central fiber can be used to slightly modify the trapping position, thus allowing to realize a particle translation or oscillation. Finally in d) a schematic representation of an opticalanalysis configuration is used. Some fibers (e.g. those with pink cores in the figure) can be used to trap the particle, while other fibers (blue cores in the example) can optically excite the sample, and the central fiber can be used for the collection of the emitted signal. It is also interesting to notice that different fibers can be used for the different tasks (e.g. a large-mode-area fiber can be used to increase signal collection)

25 Fig. 2.6: Schematic representation of different functionalities that could be obtained using the fiber-bundle TOFT. a) multiple traps realized along the probe axis by using a different cutting angle θ on three of the fibers, (b) steering of the fiber beams necessary to realize multiple traps at the same distance from the probe end c) oscillation of the trapped particle by using the light output from the central, d) optical-analysis configuration, e.g. pink core fibers can be used to trap, while blue-core ones can optically excite the sample; the central fiber can be used for the collection of the emitted signal NUMERICAL ANALYSIS The starting point of the calculation is the evaluation of the spatial distribution of the amplitude and phase of the optical field in the far field through a bidimensional Fourier transform. The limitation due to the classical paraxial approximation, which cannot be applied to strongly focused or tilted beams, has been overcame by using the angular spectrum decomposition representation [9] 9. Once the radial intensity distribution in the far field is known, the angular distribution of the rays is derived from the gradient of the optical phase, whereas the power carried by each ray is determined through the corresponding field amplitude in the far field. The frame of reference used in the calculations is shown in Figure 2.7. The axis of rotational symmetry for all the considered beams is the z-axis. Each ray is identified by three parameters: the angle φ formed 9 L. Novotny, B. Hecht, Principles of Nano-Optics, Cambridge University Press, New York, USA, 2006

26 between the ray direction and the z-axis, the azimuthal angle β, and the carried optical power. The simulations regarding a standard microscope-based OT have been performed considering a Gaussian beam characterized by a filling factor equal to 1 impinging on a high-na objective, where the filling factor is defined as the ratio between the beam waist and the objective radius [3]. The quantity NA, thus, represents, for the Gaussian beam, the value of the maximum angle between the rays and the z-axis, φ max. It is worth noticing that, by using such a large filling factor, a significant fraction of the optical power, carried by the tails of the Gaussian beam, is not collected by the objective and is consequently lost. The results presented in the following do not take into account this effect, because all the analyzed parameters are normalized with respect to the optical power that is focused on the particle, and not to the optical power input to the structure. Conversely, in the case of the TOFT, no significant power loss is present, and we calculate the quantity NA eq using the definition given in the previous section. The numerical calculation of the optical forces is performed by following the approach proposed in [3] and considering the trap geometry reported in Figure 2.7. Optical ray max y y a) z b) z o Figure 2.7: a) Ray decomposition of the optical beam: the generic ray is determined through the angles and. In the Gaussian case the angle max determines the beam NA. b) Generic ray incident on the spherical particle. For sake of simplicity in Figure 2.7 we show the case in which the center of the particle lies on the beam axis z, whereas in the following we will consider any displacement in the xyz frame of reference. The trapping beams are described through a distribution of optical rays, each of them forming an angle with respect to the beam axis. For a given, the ray is incident on the sphere forming an angle with the direction perpendicular to the surface. The total force (F T ) exerted by each ray on the particle can be obtained as the vectorial sum of the scattering and gradient component as discussed in Chapter 1.

27 2.2.3 NUMERICAL RESULTS: TRAPPING FORCES In the previous chapter we defined the optical force arising from the radiation pressure of rays composing a Gaussian beam incident on a dielectric particle in the geometric optical regime. Starting from the equations describing the behavior of the gradient and scattering force we can define the efficiency of the optical trap in the case of TOFT configuration. We start by considering a Gaussian beam tightly focused with a maximum converging angle φ max = 70, corresponding to a NA 1.25 that represents a typical situation in standard OTs, assuming water as the surrounding medium (n M = 1.33). For all the calculations reported hereafter, we will consider a wavelength λ = 1070 nm and a spherical particle with radius r = 5 μm and refractive index It is convenient to express the forces through the dimensionless Q-factor defined as Q cft n P M (20) F T is the total force exerted on the sphere and is obtained by integrating the contributions generated by all the rays composing the beam and intercepting the sphere surface. The value of Q is, thus, independent of the optical power, and it represents a figure of merit of the trapping efficacy. The behavior of Q in the yz-plane for the Gaussian beam is reported in Fig Figure 2.8: Q-factor for a Gaussian beam in standard optical tweezers. The inset shows the optical field distribution.

28 As expected, the optical forces in the case of the Gaussian beam lead to a trapping position just beyond the beam focus, and the maximum Q-value is about Let us now consider the Q-factor of an optical trap obtained by an annular core fiber through the TOFT working principle depicted in the previous section. The optical field is calculated considering that the total power is carried by the rays emitted by an annulus of diameter D = 110 μm with a beam width equal to 6 μm. Through the angular spectrum decomposition technique, it is also possible to take into account the diffraction experienced by the beam in yz-plane. The cutting angle is θ = 70 leading to a NA eq It is important to notice that such a NA eq corresponds to a converging angle φ = 45 much lower than that of the previous case. Nevertheless, as shown in Figure 2.9, a stable equilibrium of the forces is found, even if smaller Q-values (maximum Q = 0.2) are produced. Fig. 2.9: Q-factor for annular core fiber TOFT. The inset shows the optical field distribution. Considering that the structure has a cylindrical symmetry, any displacement of the sphere from the trapping point can be described using only the z-coordinate and the distance from the z-axis. As a consequence, the Q diagrams of Figures 2.8 and 2.9 can be applied to any displacement direction in the xy-plane. At last, we simulated the case of the four-fiber bundle. The optical field distribution is obtained considering that the total power carried by the annular core is now distributed over four Gaussian sources (each with mode field diameter equal to 6 μm) symmetrically disposed along the annulus, as shown in Figure 2.5(c). We consider two out of the four fibers having the axis lying in the xz-plane and the other two fibers with the axis in the yzplane. It is important to highlight that, in the bundle case, the cylindrical symmetry is broken and, differently from the previous situations, the forces depend on the displacement direction in the

29 xy-plane. The most critical trapping positions, however, are found to be in the xz- and yz-planes, where the contribution of the scattering force is most relevant. Congruently with the previous graphs, we show also in this case the Q-values in the yz-plane [Figure 2.10]; for symmetry reasons, the force distribution in the xz-plane is identical. Figure 2.10: Q-factor for a four-fiber bundle TOFT. The inset shows the optical field distribution. Also in this situation, a stable trap is formed in the beam convergence region, with values of the Q- factor similar to those of the annular case. As the total power is concentrated in the four beams, a strong gradient force is present along their propagation path, pushing the particle toward the center of each optical beam and somehow distorting the force distribution in the trapping region. A better performance could be obtained by using fibers with a larger mode size and by increasing the number of fibers included in the bundle. The obtained results confirm that, as far as the TOFT operation in the Mie regime is concerned, the fiber bundle and the annular core fiber are equivalent structures leading to similar optical force distributions NUMERICAL RESULTS: ESCAPE ENERGY In order to evaluate the trapping strength of the proposed TOFT and to compare its performances with those of the standard OT exploiting a Gaussian beam, we calculated the minimum energy, per unit power of the optical beam, necessary for a particle to escape the trap. We call this quantity ε esc. As is well known, the potential energy cannot be defined for the total optical force, as the

30 scattering component is not conservative. Hence, to find ε esc, we start considering the work per unit power (ε) that has to be done against the optical forces to move a particle from the center of the trap toward a target point (TP). The work is obtained by integrating the total force FT exerted on the sphere along linear trajectories connecting the center of the trap to TP: (21) The obtained values, in the three cases of Gaussian beam, annular cora fiber TOFT and fou-fiber bundle TOFT, are shown in Figures 2.10, 2.11 and 2.12, as a function of the TP position. Lower energy regions are indicated with a darker color. The value of ε esc, which gives a straightforward indication of the trapping strength and of the most probable escape path for the trapped particle, can be recovered by such diagrams. The meaning of ε esc can be easily understood by considering the analogous case of a potential well induced by conservative forces. A particle in a stable equilibrium point lies at the bottom of a potential well, and it can escape only if its energy is higher than the minimum energy barrier. For any possible linear escape trajectory, we evaluate the maximum value of ε: in such a way, we find a quantity ε MAX analogous to the energy barrier associated to each linear trajectory. The minimum among the calculated values of ε MAX is then simply defined as ε esc, and the corresponding trajectory is the most energetically favored escape path. As expected, for the Gaussian beam [Figure 2.11], ε esc is found considering a particle movement along the z-axis. A particle lying in the center of the trap (y = 0, z = 0) needs about 2.15 fj/w to leave the trap following the z-axis. Fig. 2.11: Work per unit power ε for bulk OT using a strongly focused Gaussian beam.

31 On the contrary, for TOFT with the annular core fiber [Fig. 2.12] we find ε esc considering as escape path the directions of propagation of the slanted rays coming from the annulus. The result is quite intuitive as, due the TOFT geometry, the scattering force is strongly suppressed along the z-axis, whereas it has a maximum along the beam propagation direction. In this case, ε esc is about 1,75 fj/w, which is slightly lower than the previously obtained value. Fig. 2.12: Work per unit power ε for annular core TOFT. Finally, we also consider the TOFT based on the fiber bundle shown in Fig We still find that the most favored escape paths are along the propagation directions of the beams emitted by the fibers, with an ε esc value similar to that found for the annular core case.

32 Fig. 2.13: Work per unit power ε for four-fiber bundle TOFT. To understand the origin of this difference in the ε esc values, Figure 2.14 compares the results obtained, for the Gaussian beam and for the annular-core-based TOFT, as a function of the NA. The values of the TOFT ε esc show a linear increase as a function of NA eq and higher values with respect to the Gaussian case. Conversely, ε esc of the Gaussian beam has a nonlinear growth characterized by an increase in the curve slope for NA > 1. This different behavior can be explained recalling that in both cases, ε esc is found along the directions where the scattering force has the maximum impact. The scattering force in the Gaussian case is essentially due to the rays on axis that carry a considerable power especially at low NA and give a negligible contribution to the gradient restoring force. On the contrary, in the TOFT annular case, the maximum scattering contribution is given by the rays coming from the annulus and strongly slanted with respect to the z-axis. As a first consequence, in the annular core case the total scattering force is not concentrated in the same z-direction, but it is distributed along the whole cone of rays. Second, the crossing-beam geometry lowers the contribution of the scattering component. It is worth noticing that Figure 2.14 highlights that the TOFT efficiency can be highly improved by increasing the device NA eq. Such a result can be easily obtained by decreasing the cutting angle θ shown in Figure 2.4(a). The TIR can still be guaranteed even for θ < θc by properly coating the fiber surface with metal. Finally, it is very interesting to analyze εesc as a function of the trapped particle radius. Fig. 2.14: ε esc calculated as a function of the NA for the focused Gaussian beam and of the NA eq for the annular core fiber TOFT.

33 2.3 FABRICATION OF A TOTAL INTERNAL REFLECTION OPTICAL FIBER TWEEZERS The TOFT fabrication process has been made in collaboration with the BIONEM laboratory of the University of Magna Graecia in Catanzaro and the Italian Institute of Technology, IIT, in Genova. It is divided in two separate steps: the first one concern the assembly of the bundle and the second regards the realization of the angled surface necessary to obtain total internal reflection FABRICATION OF THE FOUR-FIBER BUNDLE The first step of the fabrication is made in the Quantum Electronics Laboratory in Pavia. We take a bundle of four optical fibers 2 meters long with reduced cladding of 80 μm; the fibers are single mode at 1070 nm, exhibiting a mode field diameter of about 6.1 μm. First we stripe the fibers end in order to reduce their dimension, then we insert the four tips in a capillary with an internal diameter of 200 μm. We glue the fibers in position and we insert another bigger capillary with an internal diameter of 650 μm to reduce the fragility of the bundle, as schematically shown in Figure Fig. 2.15: First steps for the fabrication of a TOFT We then fill the gaps between the fibers with an epoxy resin, Epo-Tek FL, which, once solid, will held the fibers in position. In order to achieve a better penetration of the resin inside the capillary, we put the probes in a vacuum chamber for 15 minutes, and then we let the air in: the

34 air pressure will push the resin deeper inside the capillary. After three days the resin becomes solid and we can proceed with a polishing machine that will reduce the roughness of the fibers surface under 1 m, so as to obtain a good optical quality of the fiber surface. The probes are then sent to the BIONEM Laboratory in Catanzaro for the second step of the fabrication process. At the moment two different techniques have been exploited to fabricate the TOFT: in one case the cores of the fibers are cut at the desired angle to achieve total reflective by digging holes in them through a focused ion beam, in the other case prisms, having the correct angle for beam reflection, are fabricated on the surface of the fibers for the same purpose FABRICATION BY FOCUSED ION BEAM After the polishing, the probe is put in a sputtering system to deposit a thin film of gold onto the fibers surfaces. By first creating gaseous plasma and then accelerating the ions from this plasma into a gold target, the material is eroded by the hitting ions via energy transfer and is ejected in the form of neutral particles - either individual atoms, clusters of atoms or molecules. As these neutral particles are ejected, they will travel in a straight line unless they come into contact with the TOFT, coating it with a tin film of about 40 μm. A schematic representation of the sputtering technique described above is shown in Figure Fig. 2.16: Principle of the sputtering technique Once covered by a metal layer, the probe is inserted in a scanning electron microscope (SEM) with a focused ion beam (FIB) tower. While the SEM uses a focused beam of electrons to image the sample in the chamber, a FIB setup instead uses a focused beam of ions to drill holes onto the

35 sample. The FIB uses Liquid-metal ion sources (LMIS), in particular gallium ion sources. Gallium metal is placed in contact with a tungsten needle and heated. Gallium wets the tungsten and an electric field, greater than 10 8 volts per centimeter, causes ionization and field emission of the gallium atoms. Source ions are then accelerated to an energy of 5-50 kev and focused onto the sample by electrostatic lenses. LMIS produce high current density ion beams with very small energy spread. A modern FIB can deliver tens of na of current to a sample, or can image the sample with a spot size on the order of a few nanometers. Fig. 2.17: Scheme of FIB imaging As shown in Figure 2.17, the gallium (Ga+) primary ion beam hits the sample surface and sputters a small amount of material, which leaves the surface as either secondary ions or neutral atoms. The primary beam also produces secondary electrons. As the primary beam rasters on the sample surface, the signal from the sputtered ions or secondary electrons is collected to form an image. At low primary beam currents, very little material is sputtered and the FIB systems can easily achieve 5 nm imaging resolution. At higher primary beam currents, a great deal of material can be removed by sputtering, allowing precision milling of the specimen down to a sub micrometer scale. The fiber-end faces of the TOFT are then microstructured through FIB milling; the core regions at the fiber surfaces are properly shaped in such a way as to obtain TIR at the fiber core/water interface. The image [Figure 2.18] of the micromachined probe, taken at the scanning electron microscope, shows the milling of the four-fiber bundle with a trapezoidal shape, preferred to a rectangular one to minimize the damage to the sample.

36 Fig. 2.18: FIB milling steps implemented to fabricate a four-fiber bundle TOFT The overall structure presents a very good symmetry and the surfaces are of excellent quality, anyway there are some limitations. First of all there are strict conditions on the cutting angle θ L, which cannot be under 66,5 in order to have total reflection at the glass-water interface, and over 74 to obtain a gradient force high enough to trap the particle. Another problem is that the cut angle θ L cannot be perfectly achieved in the FIB process. Indeed, while the ions dig the core of the fiber at the designed angle, some of the extruded material re-deposes on the hole, thus creating heavy roughness on the core interface. Moreover the ion beam diverges as long as it goes deeper in the fiber, thus varying the cutting angle of the nucleus. Hence, the divergence of the beam and the re-deposition of the extruded material vary the angle θ L by ± 1%. The depth of the hole is also crucial, in fact we cannot verify, with the SEM vision, if the cut parameter set in the configuration exactly matches the final cut. The dig duration is quite long, it takes about half an hour for each fiber core; it is possible to reduce the time amount adding a gas etching to the ion beam, but the result is often destructive for the probe surface.

37 A big effort has been spent in increasing the performance of the optical tweezers fabrication process, in order to achieve better cuts in less time. The first implementation was the change of the dig geometry. First we define two trapezia, the smaller one superimposed to the bigger one. In this way the FIB first excavated the fiber core, obtaining the desired angle and shape, then, with the second trapezium, the dirt deposed on the cut was removed. We also switched to a bigger hole, so that the alignment between the hole and the core was less strict [Fig. 2.19]. (a) (b) Figure 2.19: Double-trapezoidal shape of the beam cut. We moved from a) 10x20x10 μm to b) 10x20x15 μm We also changed the sputtering parameters, adding a second layer of Nickel with a thickness of 80 μm on the gold one. In this way the fiber surface became more resistant to the gas etching, allowing the use of XeF 2, and reducing the drilling time without compromising the integrity of the tweezers. The different results obtained in the holes fabricated with and without the Nickel layer are shown in Figure Fig. 2.20: FIB milling with XeF 2 etching a) with Au layer and b) with Ni and Au layer

38 Finally, we set a higher beam current, which improved the fabrication time. This way we increased the number of ions hitting the probe, excavating the core more efficiently as shown in Figure Fig. 2.21: FIB milling with beam current of a) 7nA and b) 20 na After all these modification of the fabrication process, we verified the realized cutting angle and the depth of the hole. Since it has not been possible to perform this test directly on a TOFT sample, we glued two microscope slides together with a UV resin and we proceeded with the ion milling on the glass-resin-glass interfaces [Figure 2.22]. Fig. 2.22: Scheme of the process used to verify the cutting angle Then we put the glasses in a solution with acetone to remove the UV resin and we inserted one of the glasses in the SEM chamber to observe the side view of the obtained cutting angle, like that shown in Figure 2.23.

39 Fig 2.23: side-view of the FIB milling From this verification we calculated an error of ±1 on the angle, due to the material re-deposed and to the divergence of the beam. The cuts were also deeper respect to the set parameters of about 15%. Thanks to these results, we then readjusted the parameters reducing the milling time. As final result, we reduced the milling time from 26 to 14 minutes/fiber. We tried also to reduce the limitations on the cutting angle θ L, by sputtering a layer of 40 μm of Au and Ni on the fibers core of the micro-machined probe, so as to obtain total internal reflection even at lower cut angle. Anyway this coating didn t solve the problem since it was quickly burned by the radiation exiting from the cores under test FABRICATION BY TWO PHOTON LITOGRAPHY The main limitations of the FIB approach come from the fact that is very difficult, if not impossible, to obtain more complicated geometries, and from the high cost of running the FIB equipment. Two-photons lithography recently showed its ability to create microoptics of arbitrary shape on the end-face of an optical fiber.

40 The micro-prisms fabrication on each of the four fiber cores is performed by using a two-photon lithography setup where a 100-fs pulsewidth, 80-MHz Ti:sapphire laser oscillator is used as the excitation source, and a dry semi-apochromatic microscope objective, NA = 0.70, is used for beam focusing. A specific fiber holder is mounted on a xyz piezo-stage with an 80 m travel range on all axes, for positioning in horizontal and vertical directions. The fiber holder is designed in such a way to let the laser beam, coming from the microscope objective, to pass through a glass coverslip and then focalize in a photopolymerizable material droplet where the fiber bundle is immersed. A precision linear translator controls the fiber-coverslip distance. A commercial UV curing adhesive (NOA 63, Norland) is used as a photopolymer for fabrication due to its good adhesion to glass, easy processing, suitable refractive index (1.56 for the polymerized resin), and very low cost. The laser wavelength is tuned to around 720 nm and a variable attenuator, made by a half-waveplate and a polarizer, is used to decrease the laser power at the sample plane to 4 mw. The beam is expanded by a telescope, to obtain overfilling of the focusing microscope objective, and is then reflected by a 45 short-pass dichroic mirror which transmits in the visible part of the spectrum for imaging purposes. A lens images the sample, illuminated by using a light-emitting diode (LED) light source, onto a CCD camera for fiber alignment, focusing and real-time monitoring of the polymerization process. A computer-driven mechanical shutter is used to control the exposure time for each pixel. A dedicated LabVIEW-based software was written to convert the point-bypoint defined structures into piezo stage positions and to control the synchronization of the movements with the mechanical shutter. The time needed to expose a single microprism is typically around 10 min. After the completion of exposure for all of the four microprisms, the fiber bundle is retracted from the droplet and the photopolymerizable material that did not cross-link is removed by washing for a few seconds with acetone and methanol, leaving the 3-D structure attached to the fiber top. The fabricated solid 3-D microstructures are then observed by using a scanning electron microscope (SEM). In Figure 2.24 are shown the results of this technique.

41 (a) (b) Fig. 2.24: SEM images of a) the microstructured TOFT fabricated with the two-photon lithography and b) profile of the prism. 2.4 EXPERIMENTAL RESULTS This section reviews the results obtained in testing the TOFT fabricated with both the described technologies. Experiments concerning trapping and manipulation of either dielectric or biological samples are shown FIB-FABRICATED TOFT: EXPERIMENTAL RESULTS The probes have been first tested by coupling an Yb-doped fiber laser emitting at 1070 nm into the four fibers composing the bundle. The probe fibers are connected to the laser through a 1 4 fiberoptic coupler, and the optical power carried by each fiber is controlled by fiber variable optical attenuators in each path, yielding an extremely compact and stable setup, as schematically shown in Figure 2.25.

42 Fig. 2.25: Experimental setup used to test the four-fiber bundle TOFT At first we controlled that each beam focuses in the same point, thus providing the focusing effect necessary to trap particles. This experiment has been done by observing the beams with a microscope objective with high NA; the focal plane was set so as to see the fiber surfaces at first, then we observed the light distribution on focal planes with a growing distance from the fiber, the images taken during this characterization are shown in Figure Fig. 2.26: Sequence of images showing the beam focusing of the four-fibers bundle TOFT TOFT effectiveness has been successfully demonstrated by trapping water suspended polystyrene spheres (of refractive index n P = 1.59) having a diameter of 10 μm. In Figure2.27 we report a sequence of twelve images (obtained using a 10 objective) showing the TOFT and a particle that is first trapped, and then, moved under the microscope objective without escaping from the trapping position. In order to guarantee that the trapping effect was purely optical, we kept the probe at a distance of few millimeters from the cover slip, and we verified that the trapping effect vanished when the light beam was turned off.

43 Fig Sequence of images showing the movement of a trapped polystyrene bead. The experimental setup used for the trapping experiment was essentially the same used for tha beam characterization with the only exception that the TOFT was mounted on a 3-axis micromanipulator in order to move the trapped particle in a controlled manner. A final experiment has been performed to demonstrate simultaneous particle trapping and optical analysis. The probe end was immersed in a water suspension of 10 m-diameter fluorescent beads. The experimental set-up is shown in the inset of Figure By using two dichroic mirrors, either trapping or fluorescence excitation radiations (λ = 1070 nm and λ = 408 nm, respectively) were coupled into the fiber probe. The fluorescence signal emitted by the trapped bead was collected by the probe itself, transmitted by the dichroic mirrors, and then detected through a spectrometer with 10-nm spectral resolution. The optical spectra measured with and without a

44 fluorescent bead trapped by the fiber tweezers are reported in Figure The optical analysis function is very effective thanks to the short distance between the bead and the probe end, probe-end, which acts both as excitation source and signal collector. Fig. 2.28: Fluorescence experiment TWO-PHOTON LITHOGRAPHY FABRICATED TOFT: EXPERIMENTAL RESULTS By using the same experimental setup exploited for the characterization of the FIB fabricated TOFT, we evaluated the efficiency of the two-photon lithography fabricated TOFT, by trapping red blood cells and polystyrene beads. Again, we started verifying the good focusing of the four fibers, as shown in Fig

45 Fig. 2.29: Sequence of images showing the beam focusing of the four-fibers bundle TOFT Then we moved to the trapping of 10 μm polystyrene beads. To do so, we put the bundle end face parallel to the microscope slide and we pushed it inside a drop of solution with dielectric particles. After turning the laser on, we were able to trap a particle of polystyrene with all the four beams, and, as we can see in Fig. 2.30, we were able to translate the TOFT without losing the trap. (a) (b)

46 (c) (d) (e) (f) Fig : Sequence of images showing a 10 μm polystyrene beads trapped by the TOFT. In a) the scattering of the trapped particle is visible. In b)-c) we apply an IR filter to the microscope and we translate the tweezers to the risght. In d)-e)-f) we translate the tweezers in the focus plane. Then we tested thid device with a solution of red blood cells. An example of trapping is shown in Fig. 2.31: the cell is captured only by a couple of fiber, after that we switch to a four-fiber trap, and finally we continue the trapping with the second couple.

47 (a) (b) (c) (d) (e) (f)

48 (g) (h) (i) Fig. 2.31: Sequence of trapping and translation of a red blood cell. The trap is performed first by the internal prisms [a-d], then by all the four fibers [], last by the external prisms [e-f]. In i) we lost the red blood cell escape the trap. We also managed to fabricate a dual trap TOFT, cutting two couple of prisms at different angles. With this technique we put the base for multi-trap reliable fiber optical tweezers. We tested a device with the two focusing points distant 10 μm from each other in a 10 μm polystyrene beads solution. In Figure 2.32 we can see the effective trapping of two beads, while in Figure 2.33 we show a sequence of images where the polystyrene bead moves from the nearest trap to the external trap when we switch the power of the fibers.

49 Fig. 2.32: Double-trap of polystyrene beads (a) (b) Fig. 2.33: Sequence of trapping of polystyrene bead. We start by trapping the bead with the external beams (a), that create the nearest trapping point. Then (b) we switch to the second couple and the bead change position. As we demonstrated, this technique is very reliable. Respect to the other implementation, this one is cheaper and the fabrication process is faster. Moreover, thanks to the external realization of the prisms, the validation of the refraction angle is far easier and the good adhesion of the UV resin makes this technique very stable.

50 CHAPTER 3 DISCRETE ELEMENTS OPTICAL STRETCHER This chapter is composed by two main sections; one introduces the importance of cell mechanical properties in the biological field and the methods to measure them, the second one is devoted to the description of the optical stretcher which has been implemented and used to measure the elasticity of many samples. 3.1 INTRODUCTION CELL MECHANICAL PROPERTIES Cell mechanical properties are largely determined by the cytoskeleton, a polymeric network of various filaments, which plays an important role in many cellular processes. There are three different types of filaments that, together with their accessory proteins, collectively form the cytoskeleton. Actin, a semi-flexible polymer that is 7-9 nm in diameter, is made of actin protein monomers arranged in a paired helix of two protofilaments. Short actin filaments are arranged as a three-dimensional meshwork underlying the cell membrane. In addition to networks, actin can also form bundles such as stress fibers that are present between cell-substrate attachments. Microtubules are rod-like polymers of ~25 nm in diameter, and are composed of 13 protofilaments each, a linear polymer of tubulin protein subunits. They extend outward, like spokes, from the centrosome or microtubule organizing center to the actin cortex at the cell periphery. The flexible intermediate filaments are made of subunits of keratin, vimentin, desmin or neurofilament protein. They have a diameter of 8 to 12 nm, which is intermediate between that of microtubules and actin, and perform multiple roles. In one instance, they form a fibrous network that spans the cell interior and connects the nucleus to the cell membrane.

51 Figure 3.1: Cartoon of filaments composing the cytoskeleton These cytoskeletal polymer assemblies interact with themselves and with one another with the aid of several proteins such as crosslinking-, bundling- and motor-proteins. This results in a composite polymeric material that is the basic framework for various cellular activities. While cells also contain nuclei and other organelles and are surrounded by the cell membrane, these structures do not seem to contribute as much to a cell s resistance to external forces. The cytoskeleton is not only the main determinant of cell mechanics; it is also involved in many vital cellular processes. Cells expend energy to regulate their biochemical environment and actively control the conditions that lead to filament polymerization, severing, bundling, cross-linking, and sliding. In this way, the cytoskeleton is always changing and adapting to its environment. The dynamic nature of this system is critical for processes such as differentiation, mitosis, motility, intracellular transport, phagocytosis, and mechanotransduction. This link between the processes mediated by a wellregulated cytoskeleton and cellular mechanical properties can be exploited to study these processes. Whenever a cell alters its cytoskeleton, its mechanical properties change, and this can be monitored by appropriate techniques. Changes can be brought about by physiological processes, by pathological perturbations, or in response to manipulations of a researcher. While any cellular process that involves the cytoskeleton can be the target of such a study, there are some examples that seem most promising. Among physiological processes involving the cytoskeleton, the effects of mitosis on cell mechanics can be used to discriminate proliferating cells from postmitotic cells. Differentiated cells will also likely have a distinct cytoskeleton from

52 progenitor cells, which may be identified in a heterogeneous population. Likewise, motile cells such as activated macrophages can be discriminated from stationary or non activated cells. The cytoskeleton can also be modified by the addition of certain drugs and chemicals or by specific genetic modifications. Toxins such as cytochalasins, latrunculins, phalloidin, nocodazole, taxol, or bradykinin, which disrupt or stabilize specific targets in the cytoskeleton, lead to measurable changes in the physical properties of cells. Similarly, the influence of unknown chemicals, drugs, or molecules and the effect of overexpression or knockout of certain genes on the cytoskeleton can be tested by monitoring the mechanical resistance of cells. Viability tests also fall into this category, as dead cells certainly will exhibit different mechanical properties from live cells. This could be useful for drug-screening applications or for assessing transfection efficiencies. There are also many well-known examples of pathological changes that affect the mechanical properties of cells. These include cytoskeletal alterations of blood cells that cause capillary obstructions and circulatory problems; genetic disorders of intermediate filaments that lead to problems with skin, hair, liver, colon, and motor neuron diseases such as amyotrophic lateral sclerosis, and various blood diseases including malaria, sickle-cell anemia, hereditary spherocytosis, or immune hemolytic anemia. Especially well investigated is the progression of cancer where the changes include a reduction in the amounts of constituent polymers and accessory proteins, and restructuring of the cytoskeletal network, with a corresponding change in cellular mechanical properties. All of these examples suggest that mechanical properties can serve as a cell marker to investigate cellular processes, to characterize cells, and to diagnose diseases. From polymer physics we know that the mechanical strength of a network of filaments does not depend linearly on the constituent proteins. Indeed even small changes in molecular composition of the cytoskeleton and its accessory proteins are dramatically amplified in cell mechanical properties. Thus, unlike many other techniques such as Western blots, gel electrophoresis, microarrays, or FACS analysis, the measured parameter contains a built-in amplification mechanism. This benefit is accompanied by the ability to determine this parameter for single cells, not on cell populations. A few altered cells can be identified in principle against the background of many unaltered cells, leading to an excellent signal-to-noise ratio. This is especially important when only few cells are available in the first place. In addition, the intrinsic nature of the mechanical properties renders any sort of tagging preparation (radioactive or fluorescent labeling, and so on) unnecessary, saving time and cost, while leaving the cells alive, intact, and ready for further analysis or use.

53 3.1.2 EXPERIMENTAL TECHNIQUES FOR PROBING CELL MECHANICAL PROPERTIES A wide variety of experimental biophysical probes have been used to extract the mechanical properties of cells [10] 10. Figures 3.2, 3.3 and 3.4 schematically show different experimental methods used for biomechanical and biophysical probes of living cells. In particular Figure 3.2 shows: (a) atomic force microscopy (AFM), (b) magnetic twisting cytometry (MTC) and (c) instrumented depth-sensing indentation method. In these three techniques, a portion of the cell surface could be mechanically probed at forces on the order of N and displacements smaller than 1 nm. In AFM, local deformation is induced on a cell surface through physical contact with the sharp tip at the free end of a cantilever. The applied force is then estimated by calibrating the deflection of the cantilever tip, which is detected by a photodiode. MTC entails the attachment of magnetic beads to functionalized surfaces. A segment of the cell surface is deformed by the twisting moment arising from the application of a magnetic field. Elastic and viscoelastic properties of the cell membrane or sub-cellular components are then extracted from the results through appropriate analysis of deformation. Finally in the indentation test, the applied load and the depth of penetration of an indenter into the specimen are recorded and used to determine the area of contact and hence the hardness of the cell. The contact equation allows obtaining the determination of the elastic modulus of the specimen. Figure 3.2: Schematics of experimental methods used for biomechanical and biophysical probes of living cells. (a) atomic force microscopy (AFM), (b) magnetic twisting cytometry (MTC), (c) instrumented depth-sensing indentation. Figure 3.3 shows: (d) laser/optical tweezers (OT), (e) mechanical microplate stretcher (MS), (f) micro-postarray deformation (mpad) with patterned microarrays that serve as cell substrates. In 10 Biomechanics and biophysics of cancer cells, Suresh

54 these cases, forces over the range of N can be induced on the whole cell while submicrometer displacements are optically monitored. With OT, a laser beam is aimed at a high refractive index dielectric bead attached to the cell. The resulting attractive force between the bead and the laser beam pulls the bead towards the focal point of the laser trap. Two beads specifically attached to diametrically opposite ends of a cell could be trapped by two laser beams, thereby inducing relative displacements between them, and hence uniaxially stretching the cell to forces of up to several hundred piconewtons. Another variation of this method involves a single trap, with the diametrically opposite end of the cell specifically attached to a glass plate which is displaced relative to the trapped bead. In the microplate stretcher, displacement-controlled extensional or shear deformation is induced between two functionalized glass plates to the surfaces of which a cell is specifically attached. In mpad, a patterned substrate of microfabricated, flexible cantilevers is created and a cell is specifically tethered to the surfaces of these micro-posts. Deflection of these tiny cantilevers due to focal adhesions can then be used to calibrate the force of adhesion. Other patterns, such as discs and spherical islands, can also be created using micro and nano-fabrication techniques to design different substrate geometries. Figure 3.3: Schematics of experimental methods used for biomechanical and biophysical probes of living cells. (d) laser/optical tweezers (OT), (e) mechanical microplate stretcher (MS), (f) micro-postarray deformation (mpad). Finally Figure3.4 shows three others techniques: (g) micropipette aspiration (MA), (h) shear flow technique and (i) substrate stretcher. In MA, a portion of a cell or the whole cell is aspirated through a micropipette by applying suction. Observations of geometry changes along with appropriate analysis then provide the elastic and viscoelastic responses of the cell, usually by neglecting friction between the cell surface and the inside walls of the micropipette. Figure 3.4(h) instead shows a method where the biomechanical response of populations of cells could be extracted by monitoring the shear resistance of cells to fluid flow. Shear flow experiments involving laminar or turbulent flows are also commonly performed using a cone-and-

55 plate viscometer consisting of a stationary flat plate and a rotating inverted cone. Alternatively, cells could be subjected to forces from laminar flow in a parallel plate flow chamber. The mechanics of cell spreading, deformation and migration in response to imposed deformation on compliant polymeric substrates to which the cells are attached through focal adhesion complexes is illustrated schematically in Figure 3.4(i). With this technique a cell injury controller exerts a rapid positive pressure of known amplitude and duration on the substrate. The deformation of the silastic membrane, and thus the stretch of the cells growing on the membrane, is proportional to the amplitude and duration of the air pressure pulse. Figure 3.4: Schematics of experimental methods used for biomechanical and biophysical probes of living cells. (g) micropipette aspiration (MA), (h) shear flow technique and (i) substrate stretcher technique OPTICAL STRETCHER WORKING PRINCIPLE The configuration of dual-beam optical trap described in Chapter 1 is particularly interesting in the biological field because it allows either trapping in a stable and simple way any micro-particles under test, or measuring cells mechanical properties with high precision, thanks to the possibility of applying high forces in a controlled manner. To understand the origin of the forces that deform the trapped cells we can rely on the dual beam theory already discussed in Chapter 1. To simplify the trapping theory in the ray optics approach (2πr/λ<<1) we suppose that each portion of the particle surface appears as flat to the incident beams, so that we can approximate the cell with a square box with a refractive index n 2 higher than that of the surrounding medium n 1. If we consider a single Gaussian beam incident on the surface, it will carry a momentum p = n 1 E/c, where E is the energy of the beam (Minkowski form).the beam momentum is proportional to the refractive index, so it will increase while entering in the cell. We have to consider that some light is always reflected at the interface, so we have (22) where R is the reflection coefficient at normal incidence. Anyway since cells are almost transparent, the reflection is almost negligible. The momentum variation gives rise to a force that

56 tends to accelerate photons and, for Newton third law, this force is balanced by a mechanical force acting on the surface of the cell in the opposite direction, and proportional to Δp: (23) Figure 3.5: Schematics of the forces exerted by a single beam on the surface of a particle. At the second interface, as sketched in Figure 3.5, the photon exiting the particle will experience a decrease in the momentum hence the force arising on the surface cell will be directed along the propagation direction of the beam. In conclusion, when the beam passes through the particle surfaces, it stems two forces acting on those interfaces in the direction opposite to the momentum increment, thus pulling the two surfaces outwards. The second beam, entering the particle from the right as in Figure 3.6, will generate the same force contributes on the surfaces. So, by increasing power, the particle stretches. Figure 3.6: Schematics of the forces in an optical stretcher. Whereas we consider spherical particles instead of cube, we obtain a stress profile like that shown in Figure 3.7.

57 Figure 3.7: Stress profile of a spherical particle trapped in a dual beam laser The stress profile shown in Figure 3.7 is anyway an approximated result, given by the analytic function in the form: ( ) = 0 cos 2 ( ), which has been used by Guck et al. in [11] 11. In this approximation multiple reflections inside the particle have been disregarded. In literature there are more accurate approaches that define the stress profile. In his paper Chiu [12] 12 used a Ray optics (RO) approach to calculate the deforming stress acting on the surface of a cell trapped by an optical stretcher. The cells studied can be well approximated by non absorbing spheres with an isotropic index of refraction. Cells are almost transparent in the near infrared, so absorption can be neglected. The focusing power of the spherical cell concentrates the refracted rays to a smaller area on the second interface, resulting in peaks on the stress distribution around certain angular positions, as shown in Figure 3.8. Figure 3.8: Stress profile σ in Chiou approach at different distances D between the laser source and the particle [12]. 11 Stretching biological cells with light, Guck Local stress distribution on the surface of a spherical cell in an optical stretcher, Chiou 2006

58 A different approach has been proposed by Boyde et al. [13] 13. They determine the electromagnetic fields for the incidence of a monochromatic laser beam on a near-spherical dielectric particle with a complex refractive index. The perturbation approach to solve Maxwell s equations in spherical coordinates employs two alternative techniques to match the boundary conditions: an analytic approach for small particles with low eccentricity and an adapted pointmatching method for larger spheroids with higher aspect ratios. The results obtained through these calculations are shown in Figure 3.9. Figure 3.9: Time-averaged radial stresses for a spheroid in aqueous solution trapped in a double-beam laser. The laser cell distances are z0=±60 μm (left), z0= ±120 μm (middle), and z0 = ±200 μm (right). A further approach has been proposed by Nichols, [14] 14. It extends the ray-optics model by considering the focusing by the spherical interface and the effects of multiple internal reflections. Simulation results for red-blood cells (RBCs) show that internal reflections can lead to significant perturbation of the deformation, leading to a systematic error in the determination of cellular elasticity 13 Interaction of Gaussian beam with near-spherical particle: an analytic-numerical approach for assessing scattering and stresses, Boyde Determination of cell elasticity through hybrid ray optics and continuum mechanics modeling of cell deformation in the optical stretcher, Nichols 2009

59 Figure 3.9: Calculated stress distributions on a 10 μm diameter sphere trapped in an optical stretcher with a fiber separation of 200 μm. The cosine-squared approximation (dotted line) is compared to the RO model (solid line) (a) ignoring or (b) allowing for multiple internal reflections. In his paper Nichols concludes that the cosine squared angular dependence of the optical stress acting on the surface of a spherical cell can be a valid approximation to the RO stress distribution if the fiber separation is carefully chosen according to the cell radius. But this approximation also does not account for beam focusing and internal reflections that will occur within the cell that lead to regions of high optical. The inclusion of internal reflections significantly alters and reduces the range of fiber separations that would need to be selected, giving an important indication for the realization of the experiments. 3.2 EXPERIMENTAL SETUP This section is devoted to the description of the optical stretcher apparatus developed during the research activity and results obtained in the characterization of the cell elasticity OPTICAL PART The setup used to trap and stretch the cells in the dual-beam configuration in schematically shown in Figure 3.10, particularly for what concern the optical part of the apparatus.

60 Figure 3.10: Scheme of the experimental apparatus of the implemented optical stretcher. An Ytterbium doped fiber laser at a wavelength λ=1070 nm is used as source. This choice is due to the absorption spectrum of biological sample. As we can see in Figure 3.11, the window between 700 and 1100 nm presents low absorption coefficient. For higher wavelength there is a higher absorption of water, while for lower wavelength we have high absorption of melanin and hemoglobin. Fig. 3.11:Absorption spectra of cells main components

61 The use of a near infrared laser beam helps in avoiding the heating of the trapped cell, thus lowering the death probability. The beam from the laser propagates through an optical insulator, which has been introduce to block the back reflections, and is then split into two paths by an optical coupler 50%/50%. It must be noticed that the power isn t divided exactly in two, so we provided each path with a variable optical attenuator. In this way we can introduce bending power losses in order to have the same final power at the fibers tips. After the VOAs we put a couple of couplers 99%/1%; the 1% port of the first one provides a monitor that gives information about the optical power travelling in that branch, while the 1% port of the second monitor serves to check for the coupling between the two fibers, providing information about their alignment. Finally, the tips of the fibers are put on two sleighs and translated with a couple of 3-axys micromanipulators FLUIDIC PART The second part of the setup is the fluidic part that is used to deliver the cell suspension in the region where the two fibers are facing. The microfuidic system is observed with an inverted phase contrast microscope, which is very useful in biological applications because it enhances the small difference between the refractive index of water and cells; indeed with a bright field microscope it s not possible to see the cells. The images are captured by mean of a CCD camera Stingray for b/w pictures or Nikon for RBG images. DROP CONFIGURATION The first implementation of the fluidic part of the optical stretcher was very simple. It consisted in two counter propagating optical fibers having the tip inserted in a drop of solution containing the particles we wanted to analyze. Each fiber was stably put in a V-groove on an aluminum sleigh translated by a 3-axis micromanipulator.

62 Figure 3.12: Sketch of the drop configuration fluidic apparatus With this setup we have been able to trap many kinds of microparticles, either biological (stem cells, red blood cells, yeast) or non-biological (polystyrene beads, liquid crystals), as shown in Figure (a) (b) (c) (d) Figure 3.12: Examples of trapped samples. a) Polystyrene beads, b) liquid crystal cell, c) red blood cell, d) yeast organisms.

63 Anyway it has not been possible to obtain a fine stretching. The reasons are many. First there s the very interaction between fibers and the solution. A lot of particles tend to attach to the tip of the fibers, with a consequent loss of power. Moreover the translation of a fiber stems pressure forces that move the liquid, making it very challenging to trap a particle. The 3-axys micromanipulator offers 3 controlled way of freedom, but nothing can be done with rotations and we lacked a guiding structure in order to solve rotation misalignment of the fibers. Water turbulence also makes the system unstable, creating small oscillations of the fibers tip and making difficult to follow the particles. Finally the flow cannot be controlled, so it happens that more than one particle get trapped between the two fibers, preventing us from having stretching. This setup was nevertheless very useful in force measurement, as described in Appendix C. CAPILLARY-AIDED CONFIGURATION In order to get rid of some of the problems showed by the first configuration, we realized a second implementation, following the idea of optical tweezers suggested by Constable [15] 15. Our goal was to provide a guiding capillary for the optical fibers, to make an automatic alignment. The scheme is showed in Figure On a microscope slide we glued a big glass capillary, forming a smooth water-tight seal. Then we pushed two pieces of smaller capillaries along the previous one, providing a little gap for the solution drop. Finally we aligned the fibers pressing them against the couple of smaller capillaries, which formed a backstop for the fibers and provided a V groove in which they sat. This way we achieved a good alignment and we reduced the water motility thanks to the barrage of the capillary. Figure 3.14: Sketch of the capillary-aided configuration fluidic apparatus. 15 Demonstration of a fiber-optical light-force trap-constable(1993)

64 Unfortunately also this configuration showed some problems. First of all, the capillaries weren t always well aligned and, because of the bending of the fibers, we couldn t modify the alignment with micromanipulators. We lacked the translations of the fibers, so we couldn t follow the particles, we could only wait for the particles to approach the trapping area and try to remove other approaching particles. Like the previous setup we had problems with the dirt attached to the fiber-ends, which was now enhanced by the contact between fibers and microscope slide. In fact the particles suspended in the solution gradually settled on the glass, creating a layer of dirt that prevented from having a good trapping and a clear imaging (Fig. [3.15]). Fig. 3.15: Trapped polystyrene beads in capillary-aided configuration. The dirt prevents from good imaging and trapping MICROFLUIDIC CHANNEL CONFIGURATION The last implementation got rid of all the problems of the setups described. In order to obtain a microfluidic circuit to deliver the cells between the two fibers, but without inserting the fibers in the solution we followed the scheme suggested in [16] 16. We took a glass square capillary with an 16 The Optical Stretcher-A Novel Laser Tool to Micromanipulate Cells-Guck(2001)

65 internal and external dimension of 80 m and 160 m respectively and we glued it inside a couple of round capillary with internal and external diameters of 200 and 350 m respectively. Each capillary is inserted and glued in two butterfly needles, which are in turn glued to a couple of microscope slides. The two slides are connected with a couple of smaller glass rectangular capillaries, in order to have the square one suspended. A scheme and a picture of the final microfluidic circuit is shown in Figure (a) (b) Figure 3.16: (a) Scheme and (b) picture of the microfluidic circuit configuration. The solution is then inserted in a butterfly needle with a syringe and it is pushed along the microfluidic system until it reaches the other end. There are many advantages respect to the previous implementation.

66 Figure 3.17: Schemeof the flow control The optical and fluidic parts are separated, thus preventing the problem of dirt deposition on the fiber tip The flow is totally controllable, as sketched in Figure 3.17, so that we can get rid of inertial motion and we can achieve only one trapping at a time The inside of the microchannel can be cleaned, thus it is reusable The fibers facing the square capillary are aligned with a couple of 3-axys micromanipulators and the rotational misalignment can be corrected pushing the fibers until the tip touches the capillary surface. The flat interfaces between fibers, air, glass and water reduce power losses and beam deflections. Thanks to the flow control we can trap, stretch and release one cell at a time 3.3. EXPERIMENTAL RESULTS In this chapter the experimental results obtained through the microfluidic channel configuration of the optical stretcher are reported so as the automatic method used to analyze them RESULTS ANALYSIS In order to get information about the elongation of the cells we need a software able to analyze the pictures taken with the CCD [Fig. 3.18] and describe the edges of the trapped particles. For this purpose we wrote some Matlab programs, each one with a specific task.

67 Fig. 3.18: Image of a trapped red blood cell acquired with a CCD camera a) Croppa_figure.m This program loads the CCD images and asks the user to define a cropping area, in order to shrink the size of the picture [Fig. 3.19]. This helps the following analysis, reducing the elaboration time and deleting external elements that interfere with the elasticity measurement. Fig. 3.19: cropped image of a swollen red blood cell b) Analisi_multipla.m

68 Once the images are cropped around the cell, this program convert them into grayscale and exalt the contrast. Then it asks the user to point at the center of the cell and it convert the image in polar coordinates. At this point the image looks as in Fig 3.20 Fig. 3.20: Image of a red blood cell in polar coordinates Now the white annulus representing the border of the cell is stretched along the x axis. The program then asks the operator to define the area in which the border is, so the user indicates a point over the white layer and a point below it. The image is then filtered with a threshold and derivate in order to enhance the intensity gaps. Then the program finds the middle point in the white string for each column of the figure and defines a rough profile; applying an inverse Fourier transform to this line we are able to extract the dominant frequencies of the original data [Fig. 3.21a], thus obtaining a smooth profile [Fig. 3.21b].

69 (a) (b) Fig. 3.21: a) dominant frequencies and b) profile of the trapped cell The information about the x and y dimension of each cell are saved in a txt file c) Risultati_totali.m The last program loads each files txt and put all the information in an excel file. This way we get a sheet for the x and y dimension and for the ratio between the two dimensions of each cell for increasing power. Scheming the ratio x/y related to the increasing power P give the information of the cell stretching. In order to evaluate the mechanical response of the cytoskeleton, we use two different approaches. a) Step With a LabView program we set a laser power sufficient to trap the sample under test. Then we abruptly switch the laser power to a power high enough to deform the cell and with the same program we drive the CCD camera to acquire images of the trapped cells every 500 μs. We expect a deformation profile as schemed in Fig. 3.22: a high slope for few seconds after the power change, then a slow excursion till the higher deformation. This technique is useful to measure the response time of the cell to the stress.

70 RBC X dimension [μm] x/y ratio Fig. 3.22: example of a deformation profile for a step-stretching b) Ramp With another LabView program we set the initial laser power at a level sufficient for trapping. Then we slowly increase the power at regular step, taking a snapshot of the cell at every step until we reach a defined maximum value, high enough to have a sensible deformation without damaging the cell. Then we decrease the power level in the same manner. We expect a behavior like that schemed in Fig This measure is useful to measure the elasticity of the cell and to verify the viscoelastic behaviour of the deformation. X elongation x/y ratio 8 7,5 7 6, ,25 1,2 1,15 1,1 1,05 1 0, Optical power at fiber end [mw] (a) Optical power at fiber end [mw] (b) Fig 3.23: example of a) x-elongation and b) x/y ratio of a cell under ramp-stretching Not all the measurements are good for the elasticity measurement, so we have to get rid of the cells with an unexpected behavior. In fact some cells rotate when the stress is applied, or they

71 x/y ratio move vertically and change their focal plane as they become stretched. This slight change of focus can cause an apparent shift in the measured axial length and is often avoidable by increasing the trap power slightly. While rotations can usually be eliminated by good flow control, their effect on the strain curves is indistinguishable from an active behavior of the cells. Data can be improved further by removing cells whose response does not conform to a true passive viscoelastic deformation. First we can remove cells that don t respond to the applied stress. Further selectivity can be achieved by removing cells where the rate of deformation becomes negative while the stress is still being applied, and where the cell does not relax back after the stress has been reduced. These abnormal responses are likely caused by reorientation of the cell relative to the trap in addition to the deformation itself. Removing these cells from the analysis lead to a reduced ensemble of viscoelastically deforming cells. These cells are the ideal candidates to be individually fit in order to obtain physical values useful to describe their mechanical properties. Cells responses 1,15E+00 1,13E+00 1,11E+00 1,09E+00 1,07E+00 1,05E+00 1,03E+00 1,01E+00 9,90E-01 9,70E-01 9,50E Optical Power at fiber end [mw] Ideal curve movement no stretch anelastic Fig. 3.23: ideal and unexpected behaviour of cells to applied stress We applied the stretching technique to different cells; in this work we show the results of the analysis of red blood cells and tumor cells EXPERIMENTAL RESULTS ON RED BLOOD CELLS

72 Red blood cells are the simplest cells to analyze, thanks to their lack of organelles and internal nuclei. Red blood cells appear in the resting state with a biconcave shape; this is the minimum energy state. But the RBCs constantly change their shapes as they re subjected to a range of fluid forces in the circulation, as in the capillaries, where they fold along a longitudinal axis assuming an asymmetrical shape. This asymmetrical shape is maintained thanks to continuous movement of the membrane around the cytoplasm. Thus the RBC spends little of its time in discoid shape in the microcirculation. In addition, a wide variety of chemical perturbations induce shape changes, as the low concentration of fatty acids, modest changes of ph, decrement in ATP and more. In particular we ve analyzed the cytoskeleton change in red blood cells affected by three kind of diseases, in order to exploit the differences between healthy and diseased cells and create the basis for early diagnostic tests. DIABETES MELLITUS We tested the potential of the stretching technique in biological field collaborating first with the Dipartimento di Medicina interna, Istituto di cura Santa Margherita in Pavia, in order to analyze the red blood cells of geriatric patient affected by diabetes mellitus type 2; this is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Among the hemorheologic changes in the blood samples, there is an increment of the aggregation and viscosity, probably due to a decrement in the membrane elasticity. These conditions prevent the erythrocytes from adapt its shape in order to reach the smaller capillaries, thus determining the occurrence of complications. The goal of this collaboration was verifying the relation between the elasticity of the red blood cells and the status of diabetes, applying the stretching technique to healthy and diabetic patients. The RBCs were diluted in a hypotonic solution in order to swollen their shapes to simplify the elasticity analysis. Each blood sample was composed of 10 μl of blood, 4 ml of distilled water, 4 ml of physiological solution, then we added calcium albumin and glucose to provide nourish to the cells and heparin to prevent the cells from attaching to the capillaries walls. The final concentration was cells in a ml solution. Adding distilled water brings to a swollen cell, but this stress can break the RBC membrane, so that part of the Hb exits from the cell. This cause a change in the RBC refraction index, and the cell appears black with a phase contrast microscope [Fig c]. In our measurement we analyze only the healthy ones.

73 x/y ratio (a) (b) (c) Fig. 3.24:a) Biconcave red blood cell in isotonic solution. b)swollen RBC in hypotonic solution. c) Ghost RBC We analyzed the blood samples of 10 diabetic geriatric patients and 10 healthy geriatric patients. For each one we trapped and stretched 50 red blood cells, in order to get a good statistic of elongation. Each cell was stressed with a ramp approach with an optical power at fiber ends increasing from 10 to 370 mw, then decreasing back to 10 mw. The pictures were analyzed with a MatLab software in order to get information about the x and y dimensions and their ratio. These results were averaged over all the red blood cells measured for each patient. Then we averaged the results over all the patients in the same group and we compared the two groups. As shown in Fig it is clear that healthy RBCs are more elastic than diabetic ones, thus contributing to the circulation complications. 1,35 1,30 1,25 1,20 1,15 1,10 1,05 1,00 diabetic healthy Optical power at fiber end [mw]

74 Fig. 3.25: comparison in the deformation of healthy (white lines) and diabetic (blue lines) red blood cells HHT E SDS We collaborated with the Dipartimento di Patologie Umane Ereditarie in Pavia to analyze the behaviour of red blood cells affected by two kind of ereditary diseases that can bring the patient to develop anemia. These pathologies are the Hereditary Hemorrhagic Telangiectasia (HHT) and the Shwachman-Diamond Syndrome (SDS). HHT is a genetic disorder that leads to abnormal blood vessel formation in the skin, mucous membranes, and often in organs such as the lungs, liver and brain. It may lead to nosebleeds, acute and chronic digestive tract bleeding, and various problems due to the involvement of other organs. These lesions may bleed intermittently, which is rarely significant enough to be noticed but eventually leads to depletion of iron in the body, resulting in iron-deficiency anemia. SDS is a rare congenital disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities, and short stature. The most common haematological finding, neutropenia, may be intermittent or persistent and the low neutrophil counts leave patients at risk of developing severe recurrent infections that may be lifethreatening. Anemia and thrombocytopenia may also occur. Bone marrow is typically hypocellular, with maturation arrest in the myeloid lineages that give rise to neutrophils, macrophages, platelets and red blood cells. Patients may also develop progressive marrow failure or transform to acute myelogenous leukemia. Basically, both diseases can develop anemia, but for different processes. HHT causes a continuous production of red blood cells in response to the frequent bleeding, so we assume that they re younger than the cells of a healthy patient. SDS lowers the generation of red blood cells, thus they will be older than those of a healthy patient. We verified this supposition comparing the elasticity of healthy and diseased red blood cells. We prepared the same solution indicated in the diabetes experiment and we used the ramp technique to evaluate the deformation and the viscoelasticity of the samples. This work is currently under progress, but the earlier results are reassuring: as we can see in FIg. 3.25, the deformation curve of HHT patients is higher than the SDS, indicating that older cells are stiffer and demonstrating that we can distinguish the cells from each group.

75 Fig HHT and SDS Deformation EXPERIMENTAL RESULTS ON CANCER CELLS The effectiveness of the optical stretcher as a marker for biological investigation and disease diagnosis has been demonstrated by Guck and Kas in their study of tumoral cells 17 [17]. During the cell s progression from a fully mature, postmitotic state to a replicating, motile, and immortal cancerous cell, the cytoskeleton devolves from a rather ordered and rigid structure to a more irregular and compliant state. The changes include a reduction in the amount of constituent polymers and accessory proteins and a restructuring of the available network. These cytoskeletal alterations are evident because malignant cells are marked by replication and motility, both of which are inconsistent with a rigid cytoskeleton. Taken together, these changes in cytoskeletal content and structure are reflected in the overall mechanical properties of the cell as well. Thus, measuring a cell s rigidity provides information about its state and may be viewed as a new biological marker. We are collaborating with two medical groups for the analysis of tumoral cells: the CNR of Pavia for the study of fibroblasts and the IIT of Genova for the analysis of lymphocytes. LYMPHOCYTES We ve conduct experiments on three mutations of lymphocytes, analyzing their deformation under a step-stretching. The cells lines were IM9, K562 and JURKAT. IM9 [Fig. 3.26] is a lymphocytes mutation caused by multiple myeloma, a cancer of plasma cells. They cause bone lesions and they interfere with the production of normal blood cells in the bone marrow. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. 17 Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence- Guck(2005)

76 Fig. 3.26: IM9 cell K-562 [Fig. 3.27] is an erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis. Recent studies have shown the K562 blasts are multipotential, hematopoietic malignant cells that spontaneously differentiate into recognisable progenitors of the erythrocyte, granulocyte and monocytic series. K562 cells were the first human immortalised myelogenous leukaemia line to be established. The cells are non-adherent and rounded, are positive for the bcr:abl fusion gene and bear some proteomic resemblance to both undifferentiated granulocytes and erythrocytes. Fig. 3.27: K562 cell Jurkat cells [Fig. 3.28] are an immortalized line of T lymphocyte cells that are used to study acute T cell leukemia, T cell signaling, and the expression of various chemokine receptors susceptible to viral entry, particularly HIV. Their primary use is to determine the mechanism of differential susceptibility of cancers to drugs and radiation.

77 Fig. 3.28: Jurjat cell We ve performed a step-stretching of the three kind of cell starting from an initial fiber power of 35 mw and switching to 405 mw. The results are shown in the following figures. Fig. 3.29: Elongation along the x-axis of IM9 cells in a step-stretching We can see that the curve arrives at a regime value after 3 seconds. From the analysis we verified the fragility of these cells, in fact many cells break the membrane during the stretching process. Their dimensions are also very variable, they can measure from 9 to 20 μm in diameter.

78 Fig. 3.30: Elongation along the x-axis of K562 cells in a step-stretching k562 cells are more stable and resistant to stretching and thir dimension is more omogeneous, going from 13 to 18 μm. They also take 3 seconds to reach full elongation. Jurkat cells are the most resistant and the ones with a more homogenous shape. They reach the full elongation in 3 seconds and they vary from 10 to 15 μm.

79 CHAPTER 4 INTEGRATED OPTICAL STRETCHER 4.1 STRUCTURE OF AN INTEGRATED OPTICAL STRETCHER DESIGN Although the effectiveness of the OS has been widely demonstrated, the typical set-up, based on assembling optical fibers with glass capillaries or PDMS microchannels, presents some criticality mainly due to the fine and stable alignment required between discrete optical and microfluidic components. Thus the idea of developing an optical stretcher integrated on a chip made of fused silica. The lab-on-chip approach offers many advantages; it provides devices with very small dimensions, low cost and high reproducibility and it integrates microfluidic and optical functions onto a single chip. A previous work [18] 18 reported on the realization of a GaAs/AlGaAs chip for the fabrication of integrated traps exploiting a dual beam scheme. The chip, including both laser sources and microfluidic channel, has a quite complex fabrication procedure. Although efficient trapping was obtained, it should be noted that the use of semiconductor integrated lasers could reduce the chip flexibility due to the limited power available, the poor spatial quality of the optical beams and the insurgence of heating effects. In addition, the chip substrate is not transparent to visible light, thus preventing straightforward imaging of trapped cells obtainable through an optical transmission microscope. Fig. 4.1: Concept diagram showing basic implementation of Dholakia s integrated optical stretcher Recently, femtosecond lasers have been demonstrated to be valuable tools for micromachining of transparent materials; differently from standard fabrication technologies this innovative 18 S. Cran-McGreehin, T. F. Krauss, and K. Dholakia, Integrated monolithic optical manipulation, Lab Chip 6(9), (2006).

80 technique, if combined with chemical etching, is able to provide direct writing of both optical waveguides and microfluidic channels, ensuring extreme flexibility and accuracy, together with intrinsic three-dimensional capabilities. The use of femtosecond lasers for micromachining of optofluidic devices has already proved to be successful in several bio-photonic applications. Fig. 4.2: scheme of an integrated optical stretcher on a fused silica chip The integrated chip is based on a fused silica glass substrate, thus providing high transparency for cell imaging, and represents a significant improvement in terms of stability, robustness and optical damage threshold over existing optical cell stretchers. Optical trapping and manipulation of red blood cells (RBCs) in the optofluidic chip are obtained by means of two counter-propagating beams coming from two integrated optical waveguides orthogonal to the microfluidic channel. The delivery of the cell suspension to the trapping region is accomplished by an easy connection of the microchannel to an external fluidic circuit, which guarantees a controlled flow and a highthroughput analysis. A fiber laser source is butt coupled to the waveguides in the chip, delivering the light required for the trapping and stretching of cells. Since glass absorption in the wavelength

81 range adopted in the experiments (near infrared) is very low, the high powers needed for optical stretching can be easily coupled without heating appreciably the chip. Moreover, the high spatial quality of the trapping beams is guaranteed by the waveguide spatial mode distribution. The device we propose is user-friendly and reliable as it doesn t require any critical alignment between discrete optical and fluidic elements. In addition, it allows very stable and reproducible operation, which is a very important asset when quantitative analysis of the cell deformability is required SIMULATIONS In order to optimize the performance of the IOS we first perform a careful design through numerical simulations. The design variables are the distance between the waveguide end-faces and the waveguide mode size, which are in principle dependent on the size of the cells under test. In our monolithic approach such parameters are fixed once the chip is fabricated, and they must be defined in advance taking into account the target application of the device. The numerical analysis of trapping efficiency is based on the beam decomposition approach. The spatial distribution of the optical field is obtained by assuming that each waveguide emits a Gaussian beam that propagates according to paraxial approximation. Afterwards, the beams are decomposed into a set of optical rays that are defined at each propagation step along the beam propagation axis. The amplitude and the wave-front curvature of the Gaussian beams are used to assess the optical power associated to each ray and their propagation direction. Once the optical field at each position is known, the optical force exerted on a particle is calculated as the sum of the scattering and the gradient components of each ray. The total optical force distribution is then computed by summing all the contributions due to the two beams. As already introduced in Chapter 2, the effectiveness of an optical trap is evaluated through the escape energy ε esc, defined as the minimum energy needed by a particle to escape the trap, starting from its center. In order to find the value of ε esc for a specific trap, we first calculate the work (ε TP, work per power unit) that has to be done against the optical forces to move a particle along a straight line connecting the centre of the trap to any possible target point in the surrounding space. Once the ε TP distribution is known, one can determine the path energetically most favorable. The energy needed to escape the trap following such a path corresponds to ε esc. Such a parameter can be

82 adopted as a straightforward figure of merit to compare the effectiveness of a specific trap configuration. The most stable configuration will exhibit the maximum value of ε esc. We numerically optimize the device parameters by considering the trapping of spherical particles with a radius R p = 3.5 mm and refractive index n P = 1.38, surrounded by a medium with refractive index n M = We will consider red blood cells (RBCs) in hypotonic solution as experimental target samples. Indeed in such a situation the cells tend to swell, loosing their typical disk-like shape and becoming more similar to a sphere. The beam waist at the working wavelength of 1070 nm is initially set at w = 4 mm that is a value easily attainable through the fs-laser writing technology. Figure 4.3 reports the distribution of ε TP of a specific dual beam optical trap obtained considering a distance L between the waveguide end-faces equal to 150 mm. Fig. 4.3: a) Basic scheme of the optofluidic chip: the two waveguides emit counter-propagating Gaussian beams. The sample under test flows into the microchannel. L is the distance between the two waveguide end-faces; Δy indicates possible misalignment between the waveguide axes. b) Plot of the work per power unit ε TP produced by the dual beam trap. The beams (w = 4 mm) are emitted by two waveguides characterized by L = 150 mm; ε TP is expressed in fj/w. We note that a deviation of RBC shape from a sphere-like particle could lead to a slightly reduced value of the trapping stiffness. The two beams propagate along the z-axis and a perfect alignment of the two beams in the transversal direction is considered, i.e. the axes of both beams are at y = 0. As expected, ε TP behaves like a smooth potential well where the stable trapping position lies in the midpoint (z = 0) along the beam axis. Figure 4.4 reports the value of the escape energy ε esc

83 calculated for each trapping configuration obtained by changing the distance L and the transversal waveguide misalignment Δy. Figure 4.4: Contour plot of the escape energy ε esc expressed in fj/w as a function of the transversal misalignment Δy and the distance L between the waveguide end-faces. It can be easily observed that the most stable trap, corresponding to the maximum ε esc, is obtained for L = 148 mm and Δy = 0. It is worth noting that for L < 100 mm and L > 250 mm the value of ε esc becomes considerably lower and the trap cannot be considered as stable. The wide range of stable trapping is consistent with the fact that the optical beams are not focused and the trapping condition is obtained through the counterbalancing of the beams scattering components. On the other hand Figure 4.4 underlines that a transversal misalignment of Δy 1 mm already leads to a sensible variation of the trapping stiffness. The fs-laser writing fabrication procedure guarantees accuracy in the waveguides transversal position of the order of 100 nm thus preventing any criticality due to such misalignment. From the results shown in Figure 4.4 it is clear that the use of an integrated chip, where L is fixed and Δy is negligible, allows improving the reliability and the efficiency of the device. It is important to point out that the trap characteristics are dependent also on the mode size w. Anyway we found that variations of the beam waist by an amount δw = ±1 mm do not affect significantly the trapping stiffness. On the contrary, the trapping efficiency is strongly dependent on the size of the particle under test. Figure 4.5 shows ε esc for several values of R p, while keeping w = 4 mm. It is interesting to notice that ε esc presents a marked peak in correspondence of a precise value of the distance L. This behavior can be explained considering that the force applied by a Gaussian beam along the propagation axis is not a monotonic function of the distance from the

84 waist, but it has a maximum value at a position depending on the beam waist and on the particle size. From Figure 4.5 it is quite clear that once all the IOS parameters are fixed, a variation of the particle size can lead to a significant variation of the trapping conditions. For this reason different parameters should be chosen according to the size of the particles that have to be trapped in each specific application. The IOS approach is however particularly versatile; in fact, different optical traps can be monolithically fabricated across the same microchannel with different L values and this increases the range of particles that can be efficiently trapped in the same device. Fig. 4.5: ε esc as a function of the distance L between the waveguide end-faces for three different values of the radius of the particle under test. Nevertheless, considering a single waveguides pair, the analysis reported in Figure 4.5 allows choosing the set-up configuration that minimizes the effects of polydispersity that is very common in biological samples. In the case of RBCs the radius typically varies from 3 mm to 4 mm; consequently, the optimum distance between the waveguides should vary between 120 mm and 180 mm. The configuration with L = 149 mm, that is the optimum for particle with R p = 3.5 mm, guarantees the most efficient and uniform trap performance for all the RBCs under test FABRICATION The critical parameters in the fabrication of the device are the microchannel diameter, the waveguide mode size and the optical distance between the waveguide end-faces. For the microchannel diameter a value of about 100 μm is chosen, since the capillaries used in the fiber-

85 based OS have an internal dimension of the same order; the distance between the waveguide endfaces should range between 200 μm and 400 μm; the target mode size for the waveguides is set to 3.5 μm radius in order to match the fiber single mode at 1.07 μm wavelength. Indeed, in the trapping and stretching experiments a laser wavelength of λ 1 μm is chosen due to the following reasons: i) availability of compact fiber lasers with average power sufficient to achieve trapping and stretching of cells; ii) very low absorption of glass and cells; iii) possibility to filter out the laser light used for trapping, keeping the full spectrum of visible light for the cell imaging. For the fabrication of the integrated optical stretcher we used a FLICE technique, that is Femtosecond laser irradiation followed by chemical etching; it is a powerful technique able to directly fabricate buried microchannels and waveguides and to create large access holes on the side facets of the chip in order to achieve easy connection with external capillary tubes. The schematic of the set-up used for femtosecond irradiation of the sample is reported in Fig Fig. 4.6: Scheme of the experimental set-up for laser micromachining. The femtosecond laser power is controlled by a halfwave plate (λ/2 WP) and a Glan Thomson polarizer (GT POL). Second harmonic generation (SHG) is performed and the laser beam is steered by mirrors (M) to a microscope objective (OB) that focuses the fs-pulses inside the glass substrate, mounted on a computer-controlled 3D motion stage. We use the second harmonic (515 nm) of a cavity-dumped Yb:KYW oscillator providing 350-fs laser pulses at repetition rates up to 1 MHz. The laser beam is focused by a microscope objective inside the sample; the latter is translated by a computer-controlled motion stage. The glass is transparent for the used wavelength; however the high peak intensity achieved by focusing the femtosecond laser pulses induces a nonlinear absorption mechanism consisting of a combination of multiphoton absorption and avalanche ionization. The occurrence of this phenomenon is experimentally indicated by the emission of white light from the electron plasma generated at the

86 laser focus. A first consequence of the irradiation of the nanostructured glass is a slight darkening of the glass color in the modified region. This is consistent with a red shift of the absorption spectra of the glass, corresponding to a refractive index variation through a Kramers-Kronig mechanism. Moreover we have an increase in HF etching rate of fused silica, correlated to the decrease of the Si-O-Si bond angle induced by the hydrostatic pressure or compressive stress created in the irradiated region. When fused silica is irradiated, the modifications induced by the femtosecond laser pulses can be classified into three categories depending on the laser processing conditions: a) for a low fluence, a smooth modification is achieved, resulting mainly in a positive refractive index change with a very weak selectivity in etching; b) for a moderate fluence, subwavelength nanocracks are produced, yielding a high etching selectivity of the irradiated volume with respect to the pristine one (up to two orders of magnitude); c) for high fluence, a disruptive modification is obtained with the creation of voids and microexplosions. In particular, regime a) is typically suited for waveguide fabrication, while regime b) is the one employed in the first step of the FLICE technique for microchannel production. Regime c) can be used for direct laser ablation. This technique is exploited in this work to create large access holes on the side facets of the chip in order to achieve easy connection with external capillary tubes. The access-hole diameter of 350 is designed to exactly match the outer diameter of the capillary tubes; this tailoring is achieved by irradiating multiple coaxial helixes with different radii and with a pitch of 2 μm [Fig. 4.7]. The number of coaxial helixes depends on the desired size of the access hole; for a 350 μm diameter, 3 helixes are written with diameters of 80 μm, 160 μm, and 240 μm, respectively. The two access holes are connected by a straight line that, once etched, will provide a slowly tapered microchannel with a uniform central portion of 80 μm diameter where the optical trapping is achieved. The channel walls have a minimum radius of curvature of 40 μm and show the typical surface pattern obtained with this technology providing an estimated roughness in the nm range.

87 Fig. 4.7 Irradiation of access holes and round-section microchannel Irradiation is performed at 600 khz repetition rate with a pulse energy of 290 nj at the second harmonic wavelength of 515 nm. The laser polarization is set perpendicular to the microchannel axis, which is placed at a depth of 400 μm with respect to the top surface. With the highrepetition-rate laser an irradiation speed of 1 mm/s is feasible; therefore, although complex structures are irradiated, the processing of the full chip. The chip is then immersed in an ultrasonic bath with 20% of hydrofluoric acid (HF) in water for 4.5 hours to obtain the 3-mmlong buried microchannel [Fig. 4.8]. Fig 4.8: a) access holes and microchannel structures after irradiation and b) after final etching

88 Waveguide writing in the fused silica sample is performed in the same conditions used for the irradiation step in the microchannel fabrication, i.e. focusing through a 50 objective the frequency-doubled cavity-dumped Yb:KYW laser; however, this time the laser is operated at a repetition rate of 1 MHz since in this regime the fabricated waveguides exhibit lower propagation losses [Fig. 4.9]. Fig. 4.9: waveguides irradiation Waveguide writing parameters are optimized in order to have the best guiding properties at the operating wavelength of 1 μm. A pulse energy of 100 nj and a translation speed of 0.5 mm/s allows obtaining single mode waveguides at the operating wavelength with a mode intensity radius at 1/e 2 equal to ~4 μm and an ellipticity factor of 1.1. Measured propagation losses at the operating wavelength are equal to 0.9 db/cm. Multiple sets of waveguides can be fabricated on the two sides of the microchannel, with a separation between the waveguide end-faces of 100, 130, 180 and 300 μm. Each set is composed of 3 waveguides, laterally spaced by 80 μm, that are fabricated at various depths with respect to the axis of the microchannel, i.e. + 5, 0 and 5 μm. In this way different depth positions of the trap are experimentally tested. Moreover, this approach could be exploited to fabricate several parallel traps able to intercept cells flowing at different heights, thus improving the measurement throughput. So, the overall fabrication process can thus be summarized in the following steps: i) the femtosecond laser is set to a repetition rate of 600 khz and the structures for the microchannels are irradiated (typically several structures are fabricated on the same glass substrate); ii) The laser repetition rate is switched to 1 MHz without losing the alignment and the sets of waveguides are

89 written in each device; iii) the substrate is cut and different chips with 3 mm 8 mm size are obtained; iv) etching of the microchannels is performed by immersion in the HF solution. Since the irradiation of both microchannels and waveguides is performed before chemical etching, the writing of the waveguides is interrupted 500 μm before the edge of the chip, in order to avoid any etching of the regions corresponding to the waveguides. After the etching the two end-faces are polished in order to expose the waveguide input ends and perform efficient fiber coupling [Fig. 4.10]. Fig. 4.10: Microscope image of the integrated optical stretcher Once a chip is fabricated, it is connected to external fluidic and optical circuits. Using a set-up composed by an optical microscope and accurate translation stages, external capillaries are inserted in the access holes. Once the capillary is firmly inserted it is glued by a drop of UV-curable resin. The external circuit is essentially made of two butterfly needles glued to the capillaries; the tubes at the other hand of the butterfly needles act as reservoirs. Cell suspension is transported through the trapping region by a controlled microfluidic flow; this is obtained by varying the relative heights of the two reservoirs and can be finely adjusted with a micromanipulator.

90 Fig. 4.11: Connections of the integrated optical stretcher Optical fibers are aligned to the waveguides input-facets by means of two translation stages. Buttcoupling is presently used in order to have a flexible set-up, able to test all the waveguides in the chip; however, in a final device the fiber will be permanently pigtailed to the waveguide following the standard procedure developed for photonic devices in telecommunications (typical additional losses ~0.5 db).. The chip connected to the capillaries and the fibers is also glued by UV-curable resin on a thin glass slide to increase robustness of the connections but still allowing imaging of the channel content with a high magnification objective. 4.2 EXPERIMENTS The schematic diagram of the experimental set-up used to demonstrate the effectiveness of the integrated optical stretcher is shown in Fig A CW ytterbium fiber laser with an emitting

91 power up to 5W at 1070 nm, is used as light source. The beam coming from the laser is split in two branches by means of a 50%-50% fiber coupler (FC1). The optical power in each arm is then controlled by variable optical attenuators (VOAs) and monitored using the 1% port of a 99%-1% fiber coupler (FC2a); this enables to finely balance the optical power at the output of the two fibers. In order to optimize the light coupling into the chip-integrated optical waveguides, a second 99%-1% fiber coupler (FC2b) is added in the fiber line: the power coupled into one waveguide, transmitted through the microchannel and collected by the second waveguide, is thus monitored in the opposite branch. All the fiber components are single mode at the working wavelength as well as the spliced bare end-fibers. The VOAs are specified for operation up to 2 W of optical power, while we verified the FCs up to 4 W. Given the high optical threshold of the fused silica chip, the current set-up can also be used to stretch cells other than RBCs, where higher power may be needed. Fig. 4.12: experimental setup The chip is mounted on an inverted microscope equipped for phase contrast microscopy (TE2000U, Nikon). Phase contrast images of optical trapping and stretching are acquired by a CCD camera (DS-Fi1, Nikon). The pixel size for all the employed magnifications was calibrated with a grating; this allows for absolute distance measurements with a resolution of μm/pixel in the case of a 40 objective. The trapping and stretching capabilities of the chip have been tested on RBCs. The cell suspension is prepared by diluting 10 μl of blood in 8 ml of hypotonic solution in which the RBCs acquire a quasi-spherical shape with a radius of ~4 μm; the cell suspension is inserted in the microfluidic

92 circuit with a syringe. For an easy imaging of the flowing cells, the typical value of the cell speed is set in the μm/s range EXPERIMENTAL RESULTS WITH A ROUND-SECTION MICROCHANNEL First experiments are performed on the circular cross-section microchannel chip. RBCs optical trapping is achieved with an estimated optical power at each waveguide output of about 20 mw. Figure 4.13 shows a sequence of a few frames demonstrating how the trapped RBC is stable in its position even if a background flow is present (flowing cells are out of focus since they are travelling at different heights in the microchannel). Fig. 4.13: CCD sequence of frames demonstrating the optical trapping of a single RBC; solid arrow indicates the trapped cell, while dashed arrow points to an out-of-focus cell flowing below the trap. Moreover, we observe a controlled movement of the trapped RBC along the beam axis obtained by unbalancing the optical forces applied on the two sides of the dual beam trap. The force unbalance is easily achieved by varying the output power of one of the two waveguides, which can be finely tuned by adjusting the corresponding VOA [Fig. 4.14].

93 Fig. 4.14: CCD sequence of frames showing the motion of two trapped RBCs along the trap axis obtained by varying the output power of the bottom waveguide When a single cell is stably trapped in the microchannel it can be stretched along the trap axis by simultaneously increasing the optical forces applied to the cell by the two counterpropagating beams. Experimentally this is achieved by raising the emitted power from the laser source. Therefore, the trap is still stable and a progressive stretching of RBC is observed. Figure 4.15 shows a sequence of frames demonstrating the optical stretching of a single RBC. The cell can be elongated up to 25% of its initial size when increasing the waveguide output power to 300 mw. Fig. 4.15: CCD sequence of frames showing the optical stretching of a RBC from its initial shape to 25% elongation along the beam axis.

94 However, in order to achieve such a clearly visible elongation the cell is stretched into its plastic deformation regime. By stretching the cell with lower optical power smaller deformations are observed in the elastic regime, but the lens effect induced by the curvature of the microchannel prevents from a reliable retrieval of the cell contour FABRICATION OF A SQUARE-SECTION MICROCHANNEL AND EXPERIMENTAL RESULTS To solve this lens-effect problem, the square cross-section microchannel chip is used. We designed and implemented an irradiation path to obtain a square cross-section channel (SC), as shown in Figure Fig. 4.16: Sketch of the femtosecond laser beam irradiated path representing the structure that will create the microchannel with square cross-section. This is obtained by irradiating two coaxial helixes, with a pitch of 2 mm, with rectangular crosssection one inside the other. The irradiated helixes have a cross-section height and width of 45 mm and 30 mm, respectively, for the inner one and 70 mm and 60 mm, respectively, for the external one. The microchannel is then terminated by two access holes with circular cross-section that are obtained by irradiating three coaxial helixes with diameters of 60 mm, 130 mm and 200 mm, respectively, a pitch of 2 mm and a length of 800 mm per side. While in the portions of the microchannel closer to the access holes the etching smoothes out the corners of the rectangular cross-section, in the central portion of the channel, where the HF solution arrives later and acts for

95 a shorter time, the channel closely follows the irradiation path with a sharp rectangular shape (Figure 4.17). Fig. 4.17: Comparison between the fabricated microchannels with round (RC) and square (SC) cross-sections. Different sections of the channels are also shown: at the access hole entrance (SEC.AA), at the interface between access hole and the microchannel (SEC.BB), and in the center of the microchannel (SEC.CC). Irradiation is performed with a pulse energy of 300 nj and a translation speed of 1 mm/s, leading to an overall irradiation time of about 60 minutes for the complete structure. The chemical etching is executed immersing the chip in an ultrasonic bath with 20% of HF in water. A 4.5 hours etching produces the microchannel, which is 400-mm buried under the top surface, has a 2-mm length, a central rectangular cross-section of 85x75 μm, and two 800-μm-long access holes. To test this new device we first characterized the coupling losses and the trap quality of the waveguides. We evaluated the coupling losses facing couple the light in each couple of waveguides, in order to evaluate the coupling losses inside the chip when the microchannel is empty, then we do the same measurements filling the channel with the RBC suspension. The estimate losses from the left fiber to the trapped cell are in the order of 5 db in every waveguide. The trapping quality of each set of waveguides has been characterized trapping particles flowing at different velocities, then lowering the optical power of the waveguides until the particle escaped

96 from the trap. From the distance covered in a specific amount of time we measured the escape velocity of the cell at different trapping power for each set of waveguide [Fig. 4.18] Fig. 4.18: Escape velocity of a red blood cell at different trapping power in the microchannel. The dots represent the experimental measurement, the lines come from simulations. The experimental results are in good matching with the simulations. In the same way we can measure the axial forces exerted on the trapped particle by the optical forces: F 6 rv Where η is the viscosity of water, which is approximately 10-3 N s/m 2, r is the radius of the sphere, and v is the velocity. The Fig shows the relation between axial force of the trap and the power between the waveguides at different distances.

97 Fig. 4.19: Axial force exerted on a trapped particle for different power and distance of the waveguides. After the characterization we tested the stretching of red blood cells. First we trapped a red blood cell at a power of 35 mw inside the microchannel [Fig. 4.20] (a) (b) Fig. 4.20: a) trapped red blood cell and b) edge contour exploited by Matlab. Then we increased the optical power to 300 mw and, as shown in Figure 4.21, the particle stretched.

98 (a) (b) Fig. 4.21: a) stretched red blood cell and b) edge calculated with Matlab Although efficient, the square-channel microchannel brings a big problem, that is the roughness of its wall due to the etching process. This roughness can prevent from acquiring a good imaging of the sample, thus making the exploitation of the contour of the cell more difficult. Currently we re investigating possible solution, from the different concentration of hydrofluoric acid in the etching solution to the choice of another substrate or the use of laser ablation for the microchannel fabrication.