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Chapter One Introduction Optical Tweezers is a tool invented by ARTHUR ASHKIN in 1970. He was motivated by a simple calculation: using a focused beam of power 1 W striking a particle of radius of 1 wavelength we get, by conservation of momentum, a force of 10 3 dynes, assuming the particle acts as a perfect mirror reflecting all of the incident light momentum back on itself. Though it is small in absolute terms, because of small mass, the acceleration imparted is 10 5 g where g is the acceleration due to gravity! In his experiments, Ashkin observed something even more interesting. He observed that the particles were not only pushed by the radiation, they were drawn in to the axis of radiation. This was accounted to be due to intensity gradient in the laser beam. [1] This propelled the idea of optical trapping, to hold and manipulate minute objects of micron size. Soon, Ashkin and coworkers trapped the tobacco mosaic virus, certain bacteria and Escherichia Coli bacteria, [2] to show experimentally that biological objects transparent to the radiation used, could also be trapped, which led to a revolution in the micro-studies in biology. After the simple experiments on silica particles, experiments were performed demonstrating atomic beam deflection [3]. Many further experiments also revealed that atoms could also be trapped and cooled using optical tweezers [4]. Various configurations of the lasers showed that the gradient force could be used to levitate and hold micron size objects [5]. In recent experiments researchers have trapped and moved the biological cell three dimensionally [6]. They have measured elasticity of DNA by fixing one end to a substrate, other to a dielectric bead, to hold and stretch it, with help of optical Tweezers [7]. With the help of short pulsed lasers, along with the optical tweezers, holding and cutting of cell membranes has been possible, making a novel micro-surgery [8]. The amazing capability of the Optical Tweezers was shown in the experiment of measurement of motion of Kinesin Molecule 1 on a protein, which moved in nanometer steps! [9] An excellent review of all these developments using optical tweezers is given by Ashkin. [10] The experimental setup: Possible experimental setup is shown below, the actual setup prepared in this project is described in Chapter 3. Fig 1. Showing experimental setup of optical tweezers. [11] The laser light from a continuous wave source is expanded to cover largest possible part of the focusing lens 100x, 1.25 Numerical Aperture. The beam is focused onto a Polystyrene ball near the cover slip. The light of condenser from below is split into two paths, one going to Eyepiece and other to CCD for recording. The setup is basically that of inverted microscope. The setup can be modified in various ways. For position measurement of the particle, for example, a Quadrant Photo detector could be used in place of 2 CCD camera. For vibrations of a Bead attached to DNA molecule, it could be shined with another laser light and scattered light be collected on a photodiode in place of CCD camera. For advanced experiments like study of elasticity of DNA, the setup table needs to be isolated from the vibrations by using a floating table. Principles of Working: Basic principle of working of Optical tweezers is the radiation pressure. The particles receive a back kick from transmitted radiation. Hence we need material with high refractive index and transparency. Such a material bends the light more and hence causing larger change in momentum. Particles with lower relative refractive index than the medium, like air bubbles are seen to be repelled from the trap. [1] The analysis of working can be done in two ways, First, for particle size << , using electrodynamics equations to calculate the power exerted on a dielectric sphere. Second, in the regime where size >> , where ray optics holds. First case is large subject matter of [12, 13 and 14] which we will not go into here, but we will discuss the ray optic analysis to get the understanding of origin of trapping force. Qualitative as well as quantitative analysis of this was done by Ashkin and others in [15, 16]. Considering the cases when particle is in different positions relative to the focus of the laser light, analysis of forces shows that the net resultant force is always directed to this focus, showing that the trap is stable. The weakest direction of the trap is the one after focus in the direction of propagation of laser. The analysis is done by considering few prototype rays as follows: 3 Fig 2: Comparison of gradient force Vs. Scattering force. The two forces arising are due to reflected and refracted rays. The reflected rays tend to scatter the particle, the refracted rays give back kick to the particle required for trapping, and this force is the gradient force. For high numerical apertures, i.e. for high focusing of radiation, the gradient force can be made to overcome the scattering force resulting into trapping of particles, as shown in the figure above. It can also be seen to be dominant force for other positions of particle relative to focus as follows: 4 Fig 3. Force on the particle is always directed to the point of focus of the laser beam. It can be shown that the force is independent of radius of the particle in this regime, where size>>. For the Mie regime where size << , the force varies as r 3 . So for particle sizes in between, i.e. size , we expect the variation to be between r 0 and r 3 . [15] 5 The form of the force calculated by considering the geometry, numerical aperture, power and refractive index of particles is F Q . nP , where Q is a c dimensionless factor, n is the refractive index, P is the power of laser and c is the speed of light. For TEM 00 mode and numerical aperture of 1.25, Q is 0.27. For this value of Q, in the weakest direction of the trap, a spherically shaped motile living organism must exert the force of approximately 1.2 micro dynes, for power of laser 10 mW. This implies that a motile organism 10 micron in diameter which is capable of propelling itself through water at a speed of 128 micron/sec will be just able to escape the trap, in its weakest direction. [15] The trapping is more dependent on geometry than on laser power. Higher laser power can cause local heating harmful to the biological species. Trapping energies of non-motile samples are just of the order of kT, the thermal motion energy provided by the surrounding water to the specimen. 6 Chapter Two Background Since the discovery of Optical trapping by Ashkin in 1970s, there has been a spur of activity in application of optical tweezers to biological studies, as mentioned in the introduction. Following are some of the prototype examples of works experimentalists have carried all over the world, using the optical tweezer as a tool. These examples also describe the essential techniques involved in experiments using optical tweezers. I. Work on Optical Trapping and Manipulation of Viruses and Bacteria: Soon after the discovery of trapping of micron sized polystyrene latex colloidal particles and submicron size silica spheres, Ashkin and researchers experimented trapping of colloidal tobacco mosaic virus (TMV). [2] Tobacco mosaic virus is a rugged, rod like protein that traps easily and orients itself within the trap. Interesting side-benefit of this experiment was the observation that some increasing number of strange, relatively large, apparently self-propagating particles. Suspecting bacterial contamination, they combined the trap with a high resolution microscope. This confirmed the trapping of live motile bacteria and their subsequent “opticulation” (death by light). This was because of strong absorption of green light by biological species. Later experiments with Infrared yttrium/aluminum garnet laser showed drastic changes. It became possible to hold E-coli bacteria and yeast cells for hours in isolation and observe cell reproduction within the trap. Damage-free trapping of pigmented red blood cells, green plant cells, and algae was also shown. These and some more experiments on internal cell manipulation marked the beginning of the new field of optical trapping in biology. 7 The schematic of the experimental setup is as shown below: Fig 4: The 90 degree scattering from trapped particles can be viewed visually through a beam splitter (S) with a microscope (M) or recorded using a photodetector (D). [2] Main components used were: Spatially filtered Ar green laser of wavelength 5145 A0 , with power varied from 100 to 300 mW. The Gaussian beam was focused to spot diameter of 0.6microns using an objective lens of water immersion type with numerical aperture of 1.25. For identification of bacteria, microscope with 800X is used. Procedure and discussion: The sample preparation: The sample can be prepared in mono-disperse colloidal suspension in water at high concentrations. TMV is a protein of cylindrical shape, with diameter 200 A0 and length of 3200 A0 . It has negative charge in solution and an index of refraction of about 1.57. They 8 align themselves in parallel arrays in dense aqueous solutions. Somewhat diluted samples of these kind were used in the setup shown above, which was also used for trapping of silica and other particle spheres. Observation of trapping: Trapping is observed at laser powers of about 100 to 300 mW. The capture of a virus manifests itself as a sudden increase in 90 degree scattered intensity. The bacteria could be captured into the trap more easily, as they are less motile, at powers of 3-6 mW. These could be moved across transversely to capture many more bacteria. The trapping of many bacteria is possible with rapid motion in transverse direction, because the forces in transverse are much stronger than those in the axial. Also, the forward direction force in the axial direction is stronger than the reverse direction, so a rapid upward motion can result in escape of particles. At even lower powers, 1-3 mW, a different trapping mode could be observed, in which bacteria were trapped against the bottom surface of the cover slide. In this weakest direction of trapping force, the mechanical force is provided by the slide. It is still possible to move the particles transversely over the surface, because transverse forces remain quite strong even at low powers. Trapping of E-coli bacteria was even easier because of their less motility, and could be captured and manipulated rapidly on the surface and in the bulk with powers as low as fraction of an mW. Size determination: Analysis of scattered light intensity, its angular dependence gives the information about the size, shape and orientation of the trapped viruses. Polystyrene latex spheres of known size are used to calibrate the intensity vs. size. The volume computed after averaging several trapped particles, gives, an effective volume of (450 A0 ) 3 . This corresponds to a cylinder 200 A0 in diameter and 3100 A0 long, which is quite close to the volume of TMV. From this it can also be concluded that single TMV particles are being trapped. Occasional trapping of more than one virus results into a dip in photo-detector intensity. 9 Opticulation: the TMV did not suffer any changes in size, and could be trapped for several minutes without optical damage, for powers of the order of 120 mW. The reason for this stability in visible range is that these particles have strong absorption in UV. On the contrast, bacteria which could be trapped very easily at low powers of the order of few mW, were killed at 100 mW. II. Experiments in fertilization: Experiments were performed with tweezers to manipulate live sperm cells in three dimensions [18,19] and to measure their swimming forces [20]. Applications of tweezers with short pulsed lasers called “scissors” to all optical in vitro fertilization are being considered [21]. UV drilling of channels in zona ellucida of oocytes was shown to assist sperm penetration [21]. Tweezers was used to insert selected sperm into channels to effect fertilization [22, 23]. However, fertilization efficiency and questions of possible genetic damage must be further studied. Important experiments by Bern’s group measured the effects of the wavelength on optical damage processes in sperm and in other contexts using tunable Ti sapphires lasers [24]. III. Work on DNA flexibility using Backscattering from a tethered bead as a probe: For polymer based biosensors, DNA, being a good mechanically flexible molecule could be used. Shivashankar et al, [7] used optical tweezer to hold the bead attached to one end of a DNA, other end of which is attached to the glass plate. The schematic of the setup is as follows: 10 Fig 5: Inset shows Bright field image of a DNA tethered bead in the optical trap and the backscattered light image of the same bead illuminated with the red laser. Both the images are visualized using a CCD camera. Main components used for the experiment are: The optical trapping laser used is near infrared 830 nm, 150 mW maximum power laser. Infinity corrected objective lens (Zeiss Neoflar 100 X, 1.3 NA, oil immersion). Red laser to scatter light from bead, 8mW, 633 nm. Quadrant detector (UDT, Spot 4D.) Procedure and discussion: The red laser light scattered from the bead gives horizontal position of the bead using the differential output of the quadrant detector. Total sum at the quadrant detector gives the changes in the vertical position. The delicacy of the experiment lies in the precise motion of the piezoelectrically controlled stage. The minimum step size of the stage is 10 nm. 11 Force calibration is done using the Stokes drag method, by measuring the translational velocity of the stage for which a 3 micron un-tethered bead escapes from the trap. For the 50 mW trapping power, the maximum trapping force is of the order of 3.7 picoN. Initial adjustments of the quadrant detector are done using an immobile bead. It is then raised to 3 micron height. The λ-DNA used is of length 15 microns. Keeping the bead position constant, cover slip is moved in x direction to stretch the molecule to maximum length. The motion of the bead in parabolic trap of optical tweezer indicates the freedom for its vibration as constrained by the DNA molecule. So as the strand of DNA is stretched, this motion gets restricted and can be seen from the histograms of the x-y positions obtained from the quadrant detector. Since the stretching of the DNA is along the x-direction, there is asymmetry visible in the fluctuations of the bead; they are reduced in the x-direction. The observed measurements of position are found to be fitting in the theoretical analysis based on a potential for DNA. In some more experiments, by adding a protein called Rec A, which polymerizes the DNA molecule, the researchers have found that the polymerization extends the DNA molecule beyond its contour length. Thus this technique has provided a direct measurement of DNA unfolding and kinetics of binding to Rec A protein. IV. Quantitative measurements of Force and Displacement using an Optical Trap. R. M. Simmons and coworkers combined a single beam gradient optical trap with a high resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and pico-newton forces with millisecond resolution[25]. When an external force is applied to a micron sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond timescale, and thus a trapped bead 12 can be used as a force transducer. The performance was enhanced by a feedback circuit so that the position of the trap moves by means of an acousto-optic modulator to exert a force equal and opposite to the external force applied to the bead. The parameters of the trapped bead such as stiffness and response time as a function of bead diameter, bead position within the trap and laser beam power, were compared with recent ray optic calculation of the forces on trapped beads [15]. 13 Chapter Three Making of the Experimental Setup: Testing of the Biological Microscope: Schematic diagram of the Microscope used for the setup is as shown below: Eyepiece Objective Lens, could be changed from 8X to 40X and 90X Sample table Contrast Adjustment 3-D movable mirror Fig 6: Schematic diagram of the microscope. 14 Study of the functioning of microscope: The microscope has three objective lenses and one eyepiece. Any of the three objective lenses can be used to observe the sample, by rotating the holder of these lenses. The light gathered on the plane of the sample can also be varied, to adjust the contrast, using a focusing lens provided beneath the sample. The mirror has three-dimensional rotation possible, so that the place of the illuminating lamp, a tube light in this case, doesn’t affect, and the light could be directed in the sample. This facility of rotating mirror proved to be very useful when we guided the laser light and visible light alternatively into the sample. The vertical focusing of eyepiece can be adjusted with a very high precision, the least count of the motion being 2 micron. Troubleshooting the microscope: To begin with, cleaning of all the objective lenses and mirrors was done using methanol with the help of cotton plugs and tissue paper. We tested the microscope first of all with dust particles. The microscope has three magnifications, 8 X, 40X, and 90 X. The dust particles could be seen easily with 8X, but they lacked the irregularity on a micron scale, so could not be observed well under the 40X and 90X lens. So we looked out for two more samples: smeared out RBC slide, onion cells. RBCs have suitable size for the large magnifications of 40X and 90X, and could be seen as a smeared out web. While viewing the sample under 90X magnification, a problem was faced that the plane of the focus could not be reached while lowering the objective. It was realized that the sample was mounted upside down, thus RBCs were on the other side of the glass slide. This restricted viewing of RBCs using 90X, though they were seen through 40X. Upon reversal of glass slide, RBCs could be seen using 90X also. This 15 showed that 90X objective has a very small focal length, of the size of glass slide, i.e. less than 2mm. A thin layer of onion was peeled off from the skin of onion, to make a slide of onion tissue. In 8X, linear arrays of cells were seen. In 40X, few long parallelogram like onion cells were seen. In 90X, we saw one single magnified onion cell, surrounded by other. The cell walls between the cells could be clearly seen. As preparation for the further work on trapping, bacteria were observed under the same microscope. The bacteria could be observed clearly in 40X. Modification in the microscope to suit to Optical Tweezer setup: We need to inject the laser light into the objective of the microscope to focus the laser and trap the bacteria or RBCs. For this purpose, the top viewing part of the microscope was removed and laser light was injected vertically from the above. For viewing the sample, we introduced a glass slide in the path of laser light at 45 degrees, and saw the reflected light image through the eyepiece, after arranging the eyepiece at 90 degrees to the laser light. At first, it was very difficult to get the alignment of the glass slide and the eyepiece. So laser light was used from below, instead of above, to align the eyepiece. Then the laser light was replaced by usual illumination, to view the sample of smeared out RBCs slide. The slide of RBCs could be seen in 8X and 40X, though with much lower intensity and clarity than before. The RBCs could not be seen through the 90X lens. The reason turned out to be the low light gathering aperture of the 90X lens. Also the low reflection coefficient of glass, 4%, resulted in loss of image. 16 In these attempts, and in the experiments that followed, to adjust the eyepiece accurately at 90degree, and align for the reflected light, a mirror was used in place of the reflecting glass, to see image in the eyepiece. Mirror provided large reflection in comparison to glass slide, and could be used for the alignment and positioning of eyepiece very effectively. Also, the path length between the sample and eyepiece was kept more or less same as in the microscope, so as to achieve proper focusing. To overcome the problem of low reflected intensity of light, we chose to increase the intensity of illuminating light, using bulbs. The yellow bulb of 100 W got heated up very quickly, and also was too bright, and the sample would not be visible because of lack of contrast. The less intense milky bulb of 100 W also showed no contrast and the whole image used to get wiped out. Thus the option of using bulb was ruled out. It was thought of that one laser could be used to illuminate the sample and other to be focused as the Tweezer, but this too was ruled out because in the laser light, the details of sample were difficult to figure out. We tried carbon coating the glass slide to improve the reflection of image of the sample. The coating was done using the flame of a candle. This showed the images of the sample in 90X. But this method had the drawback that the laser light was totally scattered and lost its intensity after passing through the carbon coated glass slide. Hence a reduction of coating was tried out, by moving the glass slide only once over the candle. But this showed the image with less clarity and still the laser light was reduced in intensity after scattering. Finally, availability of a partially reflecting glass solved the problem of viewing the sample at the same time passing the laser light. This glass seemed to have around 10-12% reflection, and image of sample could be seen in 90X magnification also. 17 In other experiments on optical tweezers, experimentalists have used the dichromatic mirror, which reflects the illuminating wavelength, and transmits the laser light. In our setup, we didn’t need to use of a dichromatic mirror. Need of crossed polarizer: After availability of the partially reflecting glass, we used laser light to shine the sample vertically from the top. Initially we had anticipated that only the sample will reflect the laser light, and hence laser light would be very small in intensity on the eyepiece. But this assumption turned out to be wrong, and the upper part of the lens reflected large amount of light, which got reflected from the partial reflecting glass slide, and destroyed the image in the eyepiece. Hence, to cut the laser light, we used crossed polarizeranalyzer pair, polarizer immediately after the laser light and analyzer before eyepiece. This allowed the un-polarized visible light to pass through, and the laser light to get blocked. Glare of the laser light from the sample: After removing the reflection of laser light from the surface of the lens, using crossed polarizers, it was still observed that the sample with the cover slip shines back some glare of the laser light on the partially reflecting plate and hence enters in the eyepiece. This glare was removed by selectively focusing on the image in a small region of reflected light. Thus the final setup took this shape: 18 He Ne Laser Polarizer Partial Reflector Analyzer Eyepiece Fig 7: Final Setup for Optical Tweezer. 19 Chapter 4: Experimental Observations: Bacteria: A sample of bacteria subtilis was obtained from the Bio Technology Center. The bacteria were cultured overnight, before using them for the experiments. The bacteria survive at room temperatures for and hour or two, and need to be preserved in the refrigerator, where they could be stored without decay for a day. A drop of the sample of bacteria is taken on a glass slide and covered with a cover slip. The sample is then viewed under the microscope. The objective lens often touches the cover slip forcing the liquid between to move out, in which case the slide is to be again. The solution of the bacteria dries out quickly, so it has to be viewed in a short while. These bacteria are like long ropes, and they constantly keep moving in the field of view and changing their shape. They appear on the turbid background once in a while and disappear in the same, since the motion is not confined to the single plane. RBCs: Red Blood cells were isolated from a freshly collected blood sample at the school of Bio Medical Engineering. An anticoagulant solution was added to stop the blood from coagulating. The RBC sample was diluted in the saline water solution, to decrease the density of RBCs, so that they do not congregate in the field of view, and also to provide a medium for the RBCs to float around. A drop of this sample is taken on the glass slide and covered with the cover slip. For initial observations, the sample turned out to be densely packed set of RBCs. So it was further diluted to decrease the RBCs in field of view. This also allowed the motion of RBCs. But the group of RBCs moved together in a direction and slowed down until they reached the standstill, in contrast to the expectation that they would move independent of each other, due to thermal motion. 20 Both the samples have been viewed properly in 40X objective lens, but the 90X lens could not be used because of very short focal length. 21 Chapter Five: Conclusion An experiment has been set up for the optical trapping of bacteria, RBCs and other biological samples. The problem lies in viewing the samples through 90X magnification. Some other samples also need to be considered for trapping, as RBCs stick up to the glass slide and cover slip. The bacteria sample has a very small lifetime. These difficulties will be overcome by the time of presentation and the final results will be presented. This Project has introduced me to the exciting research in the field of Optical Tweezers. It has given me a very good training in literature survey, planning and development of experimental setup. 22 References: [1] First Paper: “Acceleration and Trapping of particles by radiation pressure”, A.Ashkin, Phys. Rev. Lett.1970 (156-159). [2] "Optical Trapping and Manipulation of Viruses and Bacteria", Ashkin, A.; Dziedzic, J.M., Science 235, (4795) pp 1517-20 (1987). [3] “Atomic-Beam deflection by resonance radiation pressure”, Ashkin, A. Phys. Rev. Lett., Vol.25, No. 19, 9 Nov.1970. [4] Bjorkholm, J.E. et al,(1978) Phys. Rev. Lett. 41, 1361-1364. [5] Ashkin A.,Dziedzic J.M.(1971) Appl.Phys.Lett. 19, 283-285. [6] Ashkin A.,Dziedzic J.M.(1989) Proc. Natl. Acad. Sci. USA 86.7914-7918. [7] Backscattering from a tethered bead as a probe of DNA flexibility”, Shivashankar et al, Appl. Phys. Lett. Vol. 73, No. 3, 20 July 1998, 291-293. [8] Steubing, R.W., et al, (1991) Cytometry 12, 492-496. [9] Block,S.M.,et al, (1990) Nature (London) 348,346-348. [10] Excellent Review paper: “Optical Trapping and manipulation of neutral particles using lasers”, A. Ashkin, Proc. Natl. Acad. Sci. USA Vol. 94, pp. 4853-4860, May 1997. Available online: http://www.dfi.aau.dk/~balling/alp/ashkin.pdf [11] http://atom.harvard.edu/tweezer - shows how to build a tweezer and troubleshoot it. [12] “Internal and near-surface electromagnetic fields for a spherical particle irradiated by a focused laser beam”, J.P. Barton et al, J. Appl. Phys. 64 (4), 15 August 1988. (1632-1639) [13] “Fifth order corrected electromagnetic field components for a fundamental Gaussian beam”, J.P. Barton and D.R. Alexander, J. Appl. Phys. 66(7), 1 October 1989. (2800-2802) [14] “Theoretical determination of net radiation force and torque for a spherical particle illuminated by a focused laser beam”, J.P. Barton et al, J. Appl. Phys. 66(10), 15 November 1989. (4594-4602) [15] “Forces of a single beam gradient laser trap on a dielectric sphere in the ray optics regime”, A. Ashkin, Biophys. J. Vol. 61, Feb. 1992, 569-582. 23 [16] “Observation of a single beam gradient trap force optical trap for dielectric particles”, A. Ashkin et al, Optics Lett. 11:288-290, 1986. [17] “Optical Tweezers: A new Tool for Biophysics”,Steven M.Block, Noninvasive Techniques in cell Biology:375-402 © 1990,Wiley-liss Inc. [18] Tadir, Y., et al, (1989)Fertil. Steril. 52, 870-873. [19] Colon, J. M. et al, (1992), Fertil. Steril. 57, 695-698. [20] Bonder,E.M. et al, (1990), J. Cell Biol. 111,421a. [21] Tadir,Y., et al,(1991), Hum.Reprod.6,1011-1016. [22] Schutze,K.,et al,(1994),Fertil.Steril.61,783-786. [23] Clement-Senggewald,A., et al, J.Asst.REprod.Genet.13,259-265. [24] Liang, H., et al, Biophys.J.70, 1529-1533. [25] “Quantitative Measurements of Force and Displacement Using an Optical Trap”, R. M. Simmons et al, Biophysical J., Vol.70, April 1996, 18131822. 24