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Quantification of cellular properties using ultra-high frequency single-beam acoustic tweezer
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Quantification of cellular properties using ultra-high frequency single-beam acoustic tweezer
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QUANTIFICATION OF CELLULAR PROPERTIES USING ULTRA-HIGH FREQUENCY SINGLE-BEAM ACOUSTIC TWEEZER by Hae Gyun Lim A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillments of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOMEDICAL ENGINEERING) Dec 2017 Copyright 2017 Hae Gyun Lim ii DEDICATION DEDICATED TO MY BELOVED PARENTS, MY SISTER, AND MY LOVELY WIFE iii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest appreciation for my supervisor and mentor Dr. K. Kirk Shung. He provided all his support and encouraged me with perfect guidance to achieve my Ph. D. degree. This work would not have been possible without his involvement. It was incredible to have him as my mentor. Next, I gratefully acknowledge my committee members Dr. Qifa Zhou, Dr. Keyue Shen, Dr. Jesse T. Yen, and Dr. Robert H. Chow for their insightful feedback. Also, it was my pleasure to work with the great people in the NIH Resource Center on Medical Ultrasonic Transducer Technology. My earnest thanks to all my former and current lab members Dr. Hyung Ham Kim, Dr. Jin Ho Chang, Dr. Jae Youn Hwang, Dr. Jinhyoung Park, Dr. Changyang Lee, Dr. Sangpil Yoon, Dr. Hojong Choi, Dr. Bong Jin Kang, Dr. Changhan Yoon, Dr. Mingon Kim, Hayong Jung, and Chi Woo Yoon for support throughout my research work. In addition, I am equally thankful to my colleagues Dr. Ruimin Chen, Dr. Teng Ma, Nestor E. Cabrrera-Munoz, Robert Wodnicki, Zeyu Chen, Payam Eliahoo, Xuejun Qian, Mingyue Yu, and Hsiao-Chuan Liu for their honest feedback and valuable discussions. I’d like to thank my parents Hwijong Lim and Junghi Lee. I will never be able to pay back the love you showered upon me. Once again, thank you for being my parents, and it is my great fortune being your son. You make me breathe. Also, I’d like to express my thanks to my sister, Sinyoung Lim, brother in law, Kwon Choi and niece, Yooji Choi. iv I consider myself the luckiest in the world to have such a lovely family with unconditional support. Most importantly, I owe thanks to a very special person, my wife, Yoewon Yoon for unfailing love, encouragement and support. I really value your contribution and deeply appreciate your unwavering belief in me. Words can never say how grateful I am to you. I love you from the bottom of my heart. Lastly, I’d like to acknowledge two people very special to me, my father in law and mother in law. My heart felt regard goes to them for their love and moral support. v TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix ABSTRACT ...................................................................................................................... xii CHAPTER 1 Introduction ............................................................................................... 2 1.1 General ................................................................................................................. 2 1.2 Single-Beam Acoustic Tweezers ......................................................................... 3 1.3 Ultra-High Frequency Transducers ...................................................................... 5 1.4 Calibration of Trapping Forces from SBAT at UHF ........................................... 7 1.5 Cellular Properties ................................................................................................ 8 1.6 Objective of Research ........................................................................................ 10 CHAPTER 2 Fabrication of UHF Single-Element Transducers for SBAT .................. 12 2.1 UHF Transducer for SBAT ................................................................................ 12 2.2 Design of UHF Transducers ............................................................................... 13 2.2.1 Transducer Geometry and Structure ........................................................... 13 2.2.2 KLM Modeling ........................................................................................... 14 2.3 Fabrication of UHF Transducers ........................................................................ 17 2.4 Performance Evaluation of UHF Transducers ................................................... 20 vi 2.5 Trapping Evaluation of UHF Transducers ......................................................... 24 CHAPTER 3 Calibration of Trapping Forces from SBAT at UHF .............................. 30 3.1 Background ........................................................................................................ 30 3.2 Experimental Procedure ..................................................................................... 33 3.2.1 UHF (110 MHz) Transducers Fabrication .................................................. 33 3.2.2 Micropipette Fabrication and Experimental Procedure .............................. 34 3.2.3 Calibration of Trapping Forces and Stiffness ............................................. 37 3.3 Result and Discussion ........................................................................................ 43 3.4 Concluding Remarks .......................................................................................... 49 CHAPTER 4 Quantification of Inter-Erythrocytes Forces with UHF SBAT ............... 50 4.1 Background ........................................................................................................ 50 4.2 Experimental Procedure ..................................................................................... 53 4.2.1 UHF (410 MHz) Transducers Fabrication .................................................. 53 4.2.2 Blood Sample Preparation .......................................................................... 54 4.2.3 RBC Experiments ....................................................................................... 55 4.2.4 Trapping Force Calibration using a MAT .................................................. 58 4.2.5 RBC Viability Test ..................................................................................... 61 4.3 Experimental Results .......................................................................................... 61 4.4 Discussion .......................................................................................................... 65 4.5 Concluding Remarks .......................................................................................... 68 CHAPTER 5 Investigation of Cell Mechanics using SBAT ......................................... 70 5.1 Background ........................................................................................................ 70 vii 5.2 Experimental Procedure ..................................................................................... 73 5.2.1 High Frequency (45 MHz) Transducer Fabrication .................................... 73 5.2.2 Transducer Performance ............................................................................. 75 5.2.3 Cell Preparation .......................................................................................... 78 5.2.4 Cell and Sphere Deformation by SBAT ..................................................... 79 5.2.5 Mechanical Test of Hydrogel Spheres ........................................................ 79 5.2.6 Cell Viability Test ....................................................................................... 81 5.3 Experimental Result ........................................................................................... 82 5.3.1 Deformation of Cell and Sphere under SBAT ............................................ 82 5.3.2 Mechanical Properties Measurement using MAT ...................................... 88 5.3.3 Cell Viability Test ....................................................................................... 91 5.4 Discussion .......................................................................................................... 93 5.5 Concluding Remarks .......................................................................................... 96 CHAPTER 6 Summary and Future Works ................................................................... 98 6.1 Summary ............................................................................................................ 98 6.2 Future Works .................................................................................................... 100 BIBLOGRAPHY ............................................................................................................ 103 viii LIST OF TABLES Table 2.1 Design parameters for UHF single-element transducers .................................. 15 Table 2.2 Evaluation of UHF transducers ........................................................................ 24 ix LIST OF FIGURES Figure 1.1 Schematics showing the principle of optical tweezers on ray optics (courtesy of Center of Single Molecule, University of Buffalo) ........................................ 3 Figure 1.2 The principle of the SBAT (Lee et al., 2010a). ................................................. 5 Figure 1.3 The single-element ultrasonic transducer .......................................................... 6 Figure 2.1 (a) Internal construction of an UHF single-element transducer (b) and (c) photographs of transducers at 110 MHz and 410 MHz .................................................... 14 Figure 2.2 PiezoCAD simulation results for (a) 110 MHz and (b) 410 MHz transducers ........................................................................................................................ 16 Figure 2.3 Fabrication procedures of the transducer ........................................................ 19 Figure 2.4 The pulse-echo system .................................................................................... 20 Figure 2.5 (a) Received-echo response and frequency spectrum of a 110 MHz transducer (b) and (c) Received-echo response and frequency spectrum of a 410 MHz transducer .......................................................................................................................... 22 Figure 2.6 (a) and (b) Lateral resolutions of a 110 MHz and a 410 MHz transducer. ..... 23 Figure 2.7 Schematic diagram of particle and cell manipulation ..................................... 25 Figure 2.8 A 5 µm polystyrene bead manipulation using a (a) 110 MHz and (b) 410 MHz transducer ................................................................................................................. 26 Figure 2.9 A 3.8 µm silica bead manipulation using a 410 MHz transducer ................... 27 Figure 2.10 Single RBC trapping using a 410 MHz transducer ....................................... 29 Figure 3.1 Calibration of trapping forces using a micropipette aspiration technique (a) photograph (b) schematic diagram .................................................................................... 35 Figure 3.2 SBAT and a micropipette aspiration technique on 5-µm particle ................... 37 Figure 3.3 Mesh generation and computational configuration in COMSOL ................... 41 Figure 3.4 (a) Distribution of the normalized suction pressure on the bead surface at four different displacements from the trap center (b) Side view of computational configuration (c) Simulation of force loss over displacement from the trap center with suction pressure levels, 2.0, 4.0, 6.0 and 8.0 kPa .............................................................. 43 x Figure 3.5 Relation between trapping force and displacement from the trap center for (a) different values of input voltage with PRF = 1kHz and DTF = 1% (n=5) (b) different values of DTF with PRF =1kHz, and a constant input voltage = 6.3 V PP (n=5). ................................................................................................................................. 46 Figure 4.1 Single RBC trapping with SBAT. Schematic of experimental systems. RBC was trapped by a 410 MHz transducer driven a duty factor of 1%, a PRF of 1 kHz, and a peak-to-peak input voltage 7.94 of V pp . .......................................................... 56 Figure 4.2 Schematic steps of measuring the inter RBC force. ........................................ 57 Figure 4.3 Measurement of the inter RBC forces. (a) A poly-lysine coated bead was attached to the bottom of the dish as well as a RBC aggregate. The transducer was fixed at one position and input voltage was increased starting from 0 V pp . (b-d) As the transducer gradually increased the input voltage, the RBC-2 is pulled into the acoustic trap by deforming. (e) at input voltage 7.94 V pp , the RBC-2 is completely detached from the RBC-1. (f) microscopic images of disaggregated RBCs. Red circles, yellow solid circles, and blue solid circles represent the acoustic trap zone, the center of acoustic trap and the center of RBC-2 respectfully. n=10 ................................................ 58 Figure 4.4 Calibration acoustic trapping force using a MAT. Schematic of the experimental arrangement. The RBC was trapped by a 410 MHz transducer driven at a duty factor of 1%, a PRF of 1kHz, and a peak-to-peak input voltage of 7.94 V pp . The micropipette was set up at the side of the RBC and the suction force was increased starting from 0. The RBC is moved to the tip of micropipette as the two forces, the suction force and the trapping force are balanced out. ..................................................... 59 Figure 4.5 Microscopic images of calibrating trapping force. Red circles represent the acoustic trap zone. (a) the RBC was trapped by the transducer and located 10 µm from the micropipette tip. (b-e) The suction force from the micropipette was turned on and was increased until the RBC was attached to the tip. (f) The RBC was completely attached to the micropipette tip and started to deform. (g) The RBC was completely sucked into micropipette and disappeared. ....................................................................... 60 Figure 4.6 Calibration graph for the 410 MHz transducer driven at a duty factor of 1%, a PRF of 1 kHz, and a peak-to-peak input voltage of 7.94 V pp showing the relation between the trapping force and the displacement from the trap center and the RBC center. n=10 ...................................................................................................................... 64 Figure 4.7 Trapping force effects on RBC viability. (a) Fluorescence image before trapping (control) and after trapping (10, 20, 30 min). (b) Normalized cell viability. The fluorescence intensity was normalized and compared with the control group. n=10 .................................................................................................................................. 65 xi Figure 4.8 Smaller size of ultrasonic bead than the diameter of a RBC preventing any disturbance to adjacent RBCs. .......................................................................................... 67 Figure 5.1 Fabrication of a 45 MHz transducer. (a) Receive-echo response. (b) Frequency spectrum. (c) Photograph of a 45 MHz transducer ......................................... 77 Figure 5.2 Characteristics of a 45 MHz transducer. (a) Lateral beam profile. (b) 2- dimensional I SPTA profile. ................................................................................................. 78 Figure 5.3 Schematic diagram of experimental system. The SBAT at 45 MHz was driven by sinusoidal bursts from a function generator, amplified in a 50-dB amplifier. A single cell or a single sphere was deformed due to the SBAT. ..................................... 81 Figure 5.4 Deformability of MDA-MB-231, MCF-7, and SKBR-3 cells. (a) Bright field images for each cell to show area changes before and after SBAT. Scale bars indicate 5 µm. (b) Normalized area changes of trapped cells at acoustic pressures of 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa. Error bars indicate standard deviations. Sample number for each cell line was 10. ........................................................................ 85 Figure 5.5 Deformability of 0.1, 0.3, 0.6, 0.9, 1.2% agarose hydrogel spheres. (a) Bright field images for each sphere to show area change before and after SBAT. Scale bars indicate 5 µm. (b) Normalized area changes of trapped spheres at acoustic pressures of 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa. Sample number of each concentration was 10. ........................................................................................................ 88 Figure 5.6 Mechanical test to investigate the Young’s modulus of 0.1, 0.3, 0.6, 0.9, 1.2% agarose hydrogel spheres. (a) A MAT was utilized to measure the mechanical properties of a 0.3% sphere. As suction pressure increased from the micropipette, the hydrogel sphere gradually deformed inside of the micropipette from (1) to (3). The red dotted line indicates how much the sphere was aspirated. Scale bars indicate 5 µm. (b) Measured young’s modulus for each sphere. Error bars indicate standard deviations. Sample number for each concentration was 10. ............................................. 90 Figure 5.7 Cell viability test of MDA-MB-231, MCF-7, and SKBR-3 cells. (a) fluorescence images for a MDA-MB-231, a MCF-7, and a SKBR-3 cell for 0.1% bleach test (positive control), before SBAT (control), and after SBAT (negative control). Scale bars indicate 5 µm. (b) Normalized fluorescence intensity of cells before and after SBAT. Error bars indicate standard deviations. Sample number for each cell was 10. ............................................................................................................... 93 Figure 6.1 Sketch of the particle trapping in three dimensions. The beam is focused upon polystyrene particles lying on an acoustically transparent (Baresch et al., 2016). 101 xii ABSTRACT Recently, a single-beam acoustic tweezer (SBAT) has successfully been developed for numerous biomedical applications involving trapping macromolecules and cells. Yet even with its extensive improvement, it was still difficult to apply previous ultrasonic tweezer techniques to study mechanical properties of a single cell due to the frequency limit. To overcome the previous frequency limit, fabrication of ultra-high frequency (UHF) single-element ultrasonic transducers (>100-MHz) was proposed. As these transducers’ beam width is inversely proportional to the center frequency of the transducer, the proposed transducers are able to manipulate a biological cell and a cell- sized microparticle. Also, quantitative knowledge of the trapping forces from the SBAT is necessary to allow the precise control of cell manipulation and to quantify cell mechanics. Therefore, I first calibrated the trapping forces of the UHF SBAT using a micropipette aspiration technique (MAT). As a result, calibrated trapping forces were found to exhibit a wide range, from the piconewton to nanonewton level, which is the strongest among the micromanipulation technologies. Quantification of cellular properties has been studied for cell regulation and function. I developed a method to quantify inter red blood cell (RBC) forces using SBATs with a pre-calibrated trapping force. The previous SBATs have been used to attempt to demonstrate the quantification of intercellular events but did not get meaningful results because there were limitations to frequencies. However, a newly xiii fabricated 410 MHz UHF transducer allowed the quantitative study of cell-cell interactions by producing a beam size of a few micrometers. In addition, the quantification of mechanical properties of suspended human breast cancer cells was demonstrated by a non-mechanical contact SBAT at high frequency. I obtained absolute properties of the cell membrane to determine the metastatic potential of cancer cells in vitro. Results from these examinations could demonstrate the feasibility of the SBAT, a non-destructive assessment tool of clinical diagnosis for investigating biomechanical characteristics of cells. 2 CHAPTER 1 Introduction 1.1 General In recent years, the study of intercellular forces and mechanical properties of cells has been of interest in the fields of physiology and biomedicine. This is because investigating cell mechanics could play a key role in predicting and evaluating diseases in medical and clinical fields. Various methodologies such as scanning electron microscopy (SEM) (Jan and Chien, 1973), the micropipette aspiration technique (MAT) (Buxbaum et al., 1982), atomic force microscopy (AFM) (Steffen et al., 2013), and optical tweezers (OTs) (Bronkhorst et al., 1997) have presented to quantify cell mechanics. Among them, OTs have led to the widespread dissemination of this field by allowing the precise dynamic control of nano-scale particles, viruses, and motor proteins (Mehta et al., 1999). The operating principle of OTs is based on the use of a tightly focused laser beam that is refracted on the surface of the bead. A gradient force (F G ) is generated from the change of momentum transfer from the inner region force of f a and f b , as shown in Figure 1.1. However, there are a few disadvantages of this laser use: 1. The high intensity of the laser itself may damage a biological sample by producing thermal effects on its surface area. 2. It has a shallow depth of penetration, so this technique is limited in that it cannot be applied in experiments that require penetration deeper in the medium. 3. It is difficult to monitor the phenomenon in light opaque media such as blood. 3 Figure 1.1 Schematics showing the principle of optical tweezers on ray optics (courtesy of Center of Single Molecule, University of Buffalo) The single-beam acoustic tweezer (SBAT) is an advanced technique that overcomes these drawbacks of OTs. In addition, by invoking advanced UHF ultrasonic transducer technology, the SBAT is capable of trapping a cell-sized microparticle and manipulating it. To allow the quantification of mechanical properties of various cells and molecules, calibration of trapping forces from the SBAT at UHF is proposed. Furthermore, the estimation of forces associated with cellular events using a pre- calibrated trapping force of the SBAT is demonstrated in this study. 1.2 Single-Beam Acoustic Tweezers The term, “acoustic tweezer” refers to the manipulation of a single particle or multiple particles using the ultrasonic radiation force produced by a progressive wave. Earlier versions of acoustic tweezer devices used counterpropagating waves to form a 4 beam of a standing wave, which drove particles to the pressure node. This method was used to demonstrate trapping latex spheres or frog eggs by two opposite ultrasonic transducers at 3.5 MHz (Wu, 1991). Based on the standing wave schemes, surface acoustic waves (Ding et al., 2012; Johansson et al., 2012; Tran et al., 2012) and bulk acoustic waves (Courtney et al., 2013) have been suggested as acoustic manipulation devices as well. An array transducer allowed the control of multiple particles independently using an acoustic source on one side and a reflector on the other (Courtney et al., 2014). However, due to the high demand for the simplicity of equipment and fabrication of transducers, an alternative method was necessary. Moreover, arrays are unpractical if a cell-sized microparticle manipulation is considered in the experiment because of their relatively large beam width compared to cells. Consequently, single- beam acoustical geometries (Mitri, 2013, 2014) including acoustic vortices based on a potential well (Kang and Yeh, 2010; Mitri, 2011) and an annular beam (Mitri, 2016; Mitri, 2015) for trapping have been proposed as they allow transmitting an ultrasonic beam from only one side. 5 Figure 1.2 The principle of the SBAT (Lee et al., 2010a). An analogy to OTs, Figure 1.2 demonstrates the ray acoustic regime for the SBAT. Net force (F) is generated as two represented rays (a and b) in Gaussian intensity transmitted into a sphere, and acts on the sphere as momentum transfer occurs. Based on the concept of the ray acoustic regime, when the particle size is larger than the acoustic wavelength, an SBAT with a highly focused transducer could transversely trap a particle on a membrane (Lee et al., 2009, 2010b). A 30 MHz lithium niobate transducer and a 70 MHz transducer successfully trapped a particle size of 105 µm and 90 µm, respectively (Lee et al., 2010a; Li et al., 2013) 1.3 Ultra-High Frequency Transducers An SBAT system requires a single-element ultrasonic transducer, which consists of three layers: a matching layer, a piezoelectric layer, and a backing layer. The property of the piezoelectric layer is to convert the mechanical energy to electrical energy and vice versa due to the piezoelectric effect. The matching layer increases the sensitivity by 6 reducing acoustic impedance mismatch between the piezoelectric material and the medium. The backing layer suppresses the ringing down processes of the ultrasound, resulting in a broad bandwidth. Usually, broadband transducers give improved resolution, but result in lower sensitivity. Therefore, those transducers are more suitable for imaging applications. On the other hand, narrowband transducers with greater sensitivity are more useful for the acoustic tweezer and stimulation purposes. Each transducer has its own advantages in particular applications. Figure 1.3 The single-element ultrasonic transducer One of the major current issues surrounding SBATs is developing higher frequency ultrasonic transducers. The frequency ranges of 10 to 100 MHz and over 100 MHz are designated “high frequency” and “UHF”, respectively. The main advantage of 7 higher frequency transducers is that they have a smaller beam width since their beam width is inversely proportional to the center frequency. The calculation of an f-number and the lateral resolution at the focal point of the ultrasonic beam can be predicted by (1.1~1.3): 𝜆 =𝑐/𝑓 & (1.1) 𝑓#=𝑍 ) /𝐷 (1.2) 𝑅 , =𝑓#∙𝜆 (1.3) where λ is the wavelength, c is the speed of sound, f c is the center frequency, #f is an f- number, Z f is the focal length, D is the aperture size, and R L is the lateral resolution (= the beam width). Based on those equations, smaller beam widths could be obtained by either increasing the center frequency or decreasing the f-number. Advanced UHF transducers will extend current applications of acoustic tweezers at the micrometer level to the nanometer level, which means from multiple cells to single-cell applications. Hence, they can overcome one of the current limitations on precision in ultrasound tweezer technologies. 1.4 Calibration of Trapping Forces from SBAT at UHF OTs, known as single-beam gradient traps, were first developed by A. Ashkin who discovered the phenomenon of an atomic trapping using a laser, (Ashkin et al., 1986). They became a crucial device to manipulate nano-scale particles in molecular studies. To allow the precise control of microparticles and nanoparticles, quantitative 8 knowledge of the trapping forces produced by OTs is necessary. Moreover, the estimation of forces associated with cellular events has been of significant interest. Since the physical principle of OT is similar to that of SBATs, the trapping force of the SBATs has been theoretically calculated in the same manner (Lee et al., 2013). Also, the experimental method of the drag force theorem has been employed to calibrate the trapping force of acoustic tweezers, and successfully derived the trapping force at the nanonewton level (Lee et al., 2010a). However, these previous measurement systems (Lee et al., 2010a; Li et al., 2013) are no longer applicable for calibrating the trapping force from UHF acoustic tweezers due to the much smaller size of the focal beam width as well as that of the trapped particle. A more accurate and precise method corresponding to UHF acoustic tweezers was urgently needed. An alternative method, a micropipette aspiration technique (MAT) with the advantages of reproducibility, reliability, and lower cost, was proposed to calibrate the trapping force of the SBAT at UHF. By controlling the inner size of the micropipette to be smaller than the cell size, it provides the accurate measurement of the acoustic trapping force. 1.5 Cellular Properties The properties of cells have been studied in terms of cellular functions, regulations and diseases. Among those properties, aggregation properties of RBC have especially been of significant interest since RBC aggregation affects blood circulation and tissue perfusion, including microcirculatory blood flow dynamics and venous flow 9 resistance. Abnormal RBC aggregation has been linked to diseases such as circulatory disorders, malaria, sickle cell disease (Tripette et al., 2009), and system lupus erythematosus (SLE) (Reid and De Ceulaer, 1999). Because knowledge of inter RBC forces would allow us to investigate the mechanism of red blood cell (RBC) aggregation, efforts to quantitatively measure the interactive forces of RBCs have been pursued for many years in hopes of a better understanding of hemodynamics and blood rheology. In addition, the mechanical properties of cancer cells are widely studied to learn their effects on various cellular functions such as proliferation, migration, and differentiation (Deguchi and Sato, 2009). In particular, the mechanical stiffness level of cancer cells provided the determination of the invasion potential of the cancer cell and it was reported that highly invasive cancer cells have softer membrane characteristics than weakly invasive ones (Swaminathan et al., 2011). Investigating cellular mechanical properties could play a key role in predicting and evaluating such diseases in medical and clinical fields. In order to predict those properties of cells, I proposed the SBAT as a tool of force transducers to quantify inter RBC forces and the deformability of the cancer cell at a single cell level. As mentioned above, various methodologies such as SEM, AFM, the MAT, OTs, and SBATs have been applied to characterize fundamental properties of cells. Each method has its own strengths and weaknesses. The SBAT has advantages over other technologies in cell applications: 1. It produces stronger trapping forces with a wide range. 2. The object is not required to be labeled. 3. It allows non-mechanical contact with the surface of the object. In the near future, with those efforts on qualification of the 10 properties of cells, SBAT technologies may be viable tool to identify biomarkers for detecting cell diseases. 1.6 Objective of Research The overall objective is to experimentally demonstrate the quantitative measurement of intercellular and mechanical properties of a cell using the SBAT. This thesis is composed of six chapters. In chapter 1, the general idea and background knowledge of acoustic tweezers, single-element transducers, calibration of trapping forces, and cellular properties is described. In chapter 2, fabrication of single-element transducers at 110 MHz and 410 MHz, and their performance evaluations are demonstrated. In chapter 3, calibration of trapping forces from SBATs is illustrated. In order to calibrate the trapping force from SBAT at UHF, a new calibration method, a MAT is used. The trapping force is calculated against the suction force from a micropipette. Moreover, a trap stiffness of an elastic spring at the focal zone is calculated based on a plot of “trapping force – displacement from the trap center”. Chapter 4 describes the feasibility of the SBAT to measure the inter-erythrocyte forces. Under the SBAT at UHF, I successfully trap and manipulate a single RBC, detaching it from its aggregation. Inter RBC forces is measured against a pre-calibrated trapping force using a MAT. In chapter 5, I investigated the cancer cell mechanics using the SBAT. High frequency SBAT traps and deforms a cell or a cell-mimic sphere to measure the mechanical properties of a cell. The deformability of a cell is calibrated by developing cell-mimicking spheres with the known Young’s modulus. This enables us to indirectly 11 quantify the absolute mechanical properties of cells. Finally, chapter 6 summarizes all the results in this research and explains future work. 12 CHAPTER 2 Fabrication of UHF Single-Element Transducers for SBAT 2.1 UHF Transducer for SBAT A single-beam acoustic tweezer (SBAT) is a novel tool for trapping an object, and efforts on the development of higher frequency transducers have been devoted for trapping a smaller object. Earlier studies were carried out with transducers at frequencies under 50 MHz, and were only capable of trapping a particle in the range of hundreds of micrometers. In order to carry out more practical applications of using a particle at the cellular-level, the particle size should be reduced to a few micrometers. Recently, many cellular studies have been performed using higher frequency transducers (Hwang et al., 2016; Hwang et al., 2014b). However, since the transducer’s beam width was still larger than the cell size, ultrasonic radiation forces could cause the significant disturbance to the adjacent cells during measurements of intercellular forces. In order to measure inter cohesive forces between cells such as RBCs (diameter 7~8 µm), it was necessary to develop even higher frequency transducers with the focal size smaller than the cell size. Therefore, I proposed to fabricate UHF transducers to overcome previous limitations allowing more effective trapping of a single cell and a cell-sized particle. 13 2.2 Design of UHF Transducers 2.2.1 Transducer Geometry and Structure UHF single-element transducers allow the precise control of a cell with a narrow lateral resolution at the focal point. Since the wavelength of 410 MHz transducers is only 3.5 µm, the ultrasonic wave can be tightly focused within a cell to trap. I designed and fabricated a 110 MHz and a 410 MHz single-element transducer. Figure 2.1 (a) shows the internal construction of UHF single-element transducer, and Figure 2.1 (b) and (c) are photographs of fabricated transducers. (a) 14 (b) (c) Figure 2.1 (a) Internal construction of an UHF single-element transducer (b) and (c) photographs of transducers at 110 MHz and 410 MHz 2.2.2 KLM Modeling The 36° rotated Y-cut lithium niobate (LiNbO 3 ) single crystal was purchased from Boston Piezo-Optics (Bellingham, MA, USA). LiNbO 3 is preferred for the design of a large aperture device and also offers a high sensitivity due to its desirable properties of a good electromechanical coupling coefficient (k t ~0.49), a low dielectric permittivity 15 (ε s ~39), and a high longitudinal sound velocity (c~7340 m/s). The optimized aperture size and thickness of the LiNbO 3, and the parylene matching layer thickness were designed with the Krimholtz, Leedom, and Matthaei model (PiezoCAD, Sonic Concepts, Bothell, WA, USA). Table 2.1 specifically provides the thickness of materials for each targeting frequency. Figure 2.2 is the simulated frequency spectrums for transducers at 110 MHz and 410 MHz. Table 2.1 Design parameters for UHF single-element transducers Layer Material Thickness (µm) 118 MHz 410 MHz Piezo LiNbO 3 25 7.1 Matching layer Parylene 5 1.1 Backing E-Solder 3022 1000 1000 16 (a) (b) Figure 2.2 PiezoCAD simulation results for (a) 110 MHz and (b) 410 MHz transducers 17 2.3 Fabrication of UHF Transducers Following steps were taken to fabricate a 410 MHz LiNbO 3 press focused ultrasonic transducer. The backside of the LiNbO 3 was sputtered with a 1500 angstrom (Å) chrome and gold layer. A conductive silver epoxy (E-SOLDER 3022, Von Roll Isola, USA) backing layer with an acoustic impedance of 6 MRayl was casted onto the backside of LiNbO 3 , centrifuged at 3000 rpm for 15 min, and cured overnight at room temperature. The acoustic stack including the piezoelectric material and the backing layer was pasted on the glass with wax. The backing layer was lapped down to its designed thickness of 1 mm first and then, LiNbO 3 was lapped down to 7.1 µm with an extreme care, and diced into the size of 250 µm x 250 µm using a dicing saw (Tcar 864-1, Thermocarbon, Casselberry, FL). It should be noted that the thickness of materials is the most important factor to determine the center frequency. It was a challenging and time consuming process since manually lapping down to a few micrometers requires extremely precise control. The acoustic stack was placed in a brass housing concentrically, and the gap between the stack and the housing was filled by an insulating epoxy (Epo-Tek 301, Epoxy Technologies, Billerica, MA). A single silver coated copper wire was connected to the backing layer using conductive silver epoxy. The transducer was assembled in a high frequency Sub Miniature version A (SMA) electrical connector. Mechanical press focusing was performed using a heated bearing ball to obtain an f-number 1.3. This bearing ball of 0.6 mm diameter was highly polished to make a smooth surface of the LiNbO 3 . The electrodes were sputtered with a layer 1500Å chrome and gold layer on the front surface of the piezoelectric layer to make the ground connection between the 18 elements and the brass housing. A 1.1 µm thick parylene layer was vapor-deposited on the front face of the transducer using a PDS 2010 Labcoater (SCS, Indianapolis, IN). This parylene coating served as the matching layer as well as a protection layer. Figure 2.3 shows fabrication procedures as listed: 1. Attaching LiNbO 3 to a glass plate 2. Lapping down LiNbO 3 3. Sputtering LiNbO 3 4. Bonding to the backing layer 5. Dicing 6. Fabrication of housing brass 7. Connecting the wire 8. Press focusing 9. Parylene coating and 10. Final product. Fabrication procedure of 110 MHz transducers is similar with that of 410 MHz transducers, but the aperture size and the thickness of the piezoelectric material and the matching material thickness are different. LiNbO 3 was lapped down to 25 µm with extreme care, and diced into the size of 700 µm x 700 µm using a dicing saw. A 5 µm thick parylene layer was vapor-deposited using a PDS 2010 Labcoater. A 1.8 mm bearing ball was used for press-focusing. 19 Figure 2.3 Fabrication procedures of the transducer 20 2.4 Performance Evaluation of UHF Transducers One-way sound field measurement using a hydrophone is only available with transducer at frequencies less than 60 MHz. Due to the unavailability of a practical device to evaluate the performance of UHF transducers, I performed a two-way pulse- echo test. Figure 2.4 shows the pulse-echo system. Transducers were connected to pulser/receiver, a JSR (Pittsford, NY, USA) model and excited by electrical impulses at 500 Hz at 50 damping. The targeting reflector was quartz. Figure 2.4 The pulse-echo system 21 (a) (b) 0.9 0.92 0.94 0.96 0.98 1 −2 −1 0 1 2 Amplitude (V) Distance (mm) 50 100 150 200 −10 −8 −6 −4 −2 0 Frequency (MHz) Amplitude (db) 52.04%→ ← 22 (c) Figure 2.5 (a) Received-echo response and frequency spectrum of a 110 MHz transducer (b) and (c) Received-echo response and frequency spectrum of a 410 MHz transducer Figure 2.5 displays receive–echo responses and frequency spectrums of fabricated transducers. Measured results are described in Table 2.2. The lateral resolution was experimentally measured with a 2.5 µm tungsten wire (California Fine Wire, Grover Beach, CA, USA) scanned in the lateral direction at a resolution of about 0.5 µm. The received signal reflected from the wire was analyzed to generate acoustic intensity at frequency. Figure 2.7 shows the relative magnitude as a function of lateral distance for 110 MHz and 410 MHz transducers. Measured results are described in Table 2.2. 23 (a) (b) Figure 2.6 (a) and (b) Lateral resolutions of a 110 MHz and a 410 MHz transducer. 24 Table 2.2 Evaluation of UHF transducers 110 MHz 410 MHz Center frequency (MHz) 110 410 -6dB fractional bandwidth (%) 52 31 Focal point (mm) 0.900 0.324 F-number 1.3 1.3 -6 dB lateral resolution (µm) 20 6.5 2.5 Trapping Evaluation of UHF Transducers Two-dimensional acoustic tweezer manipulation of a single microparticle and a RBC was demonstrated by 110 MHz and 410 MHz transducers. These UHF transducers with a low f-number transducer allowed the precise manipulation of a cell-sized object. The experimental arrangement is illustrated in Figure 2.7. A sinusoidal burst signal generated by a function generator (Stanford Research Systems, Sunnyvale, CA) and amplified by a 50-dB power amplifier (525LA, ENI, Rochester, USA) was used to drive the transducers. The trapped object was manipulated via a three axis motorized linear stage (SGSP 20, Sigma KOKI Co., Japan). Driving conditions are a duty factor of 1%, a pulse repetition frequency (PRF) of 1kHz, a driving voltage of 7.9 V PP , and an excitation frequency of 110 MHz and 410 MHz each. The motion of a trapped sample was observed using an inverted microscope (Olympus IX-71, Center Valley, PA), and recorded via a CMOS camera (ORCA-Flash2.8, Hamamatsu, Japan). 25 Figure 2.7 Schematic diagram of particle and cell manipulation A mobilized cell and bead were monitored using bright-field imaging. Figure 2.8 shows the trapping performance of a 5 µm polystyrene bead (Polysciences Inc., Warrington, PA) using (a) 110 MHz and (b) 410 MHz transducers. Figure 2.9 shows a 3.8 µm silica bead trapped by 410 MHz transducers. Black circles represent the initial location of the bead while the arrow is given the reference of the transducer movement. In previous studies, both a polystyrene and a silica microparticle have been used for various cellular applications to lead the cell membrane deformation or elevate chemical reactions (Fernandes et al., 2013; Hénon et al., 1999; Lenormand et al., 2001). 26 (a) (b) Figure 2.8 A 5 µm polystyrene bead manipulation using a (a) 110 MHz and (b) 410 MHz transducer 1 2 3 4 25#µm 27 Figure 2.9 A 3.8 µm silica bead manipulation using a 410 MHz transducer 1 2 3 5"µm 28 A single RBC (diameter ~7.5 µm), smaller than a normal cell (diameter ~20 µm) was manipulated by a 410 MHz transducer as shown in Figure 2.10. RBC is a non- spherical cell, which has not been previously trapped by any acoustic device due to its concave shape. Moreover, previous models of the SBAT could only manipulate a particle of a few hundred of micrometers due to the limitation of the low lateral resolution of transducers. In this chapter, I fabricated advanced UHF single-element transducers with low f-number (1.3) to improve their lateral resolution allowing single cell manipulation. As a 410 MHz transducer generated a tight focused ultrasonic beam (~6.5- µm) within a RBC, the RBC could be successfully trapped and manipulated. In chapter 4, RBC trapping with the SBAT was used for investigating the inter RBC forces. 29 Figure 2.10 Single RBC trapping using a 410 MHz transducer 30 CHAPTER 3 Calibration of Trapping Forces from SBAT at UHF *In this chapter, a substantial portion of the original paper including figure 3.1~3.4 was reprinted with permission from H. G. Lim, Y. Li, M. Lin, C. Yoon, C. Lee, H. Jung, R. H. Chow, and K. K. Shung, “Calibration of trapping force on cell-size objects from ultrahigh-frequency single-beam acoustic tweezer”, IEEE Trans. Ultrasonics Ferroelectr. Freq. Contr., vol. 63, no. 11, pp. 1988-1995, 2016 (© [2016] IEEE). 3.1 Background Acoustic tweezers use the ultrasonic radiation force to manipulate a single particle or multiple particles in applications of bioengineering (Hu and Santoso, 2004; Lee et al., 2009; Wu, 1991). More precisely, they have been used to trap and move the cells and organisms without contact in a controlled environment from water to body fluids in the field of biomedicine (Ding et al., 2012; Evander et al., 2007). Among acoustic tweezer technologies, single-beam acoustic tweezers have been getting more issue because of its simplicity of equipment setup and a transducer design by transmitting the ultrasonic beam from only one side. A single-beam demonstrated simultaneous multi-trapping of microparticles at different points (Silva and Baggio, 2015) and showed the capability of levitating one or more large particles in three dimensions using the gradient force (Baresch et al., 2016). However, the trapped particle size is quite large in a range of a few hundred micrometers because applied frequencies on their experiments were in the low MHz range. In order to trap a smaller particle, the highly focused single-beam acoustic tweezer (SBAT) based on the concept of the ray acoustic regime where particle size is 31 larger than the acoustic wavelength has been proposed, (Lee et al., 2013). As the SBAT technology reaches 200 MHz or higher, the beam size approaches to the cellular-level and it allows trapping a single cell or a cell-sized microparticle. Furthermore, an UHF transducer over 400 MHz could generate the acoustic beam smaller than RBC (~diameter of 7.5 µm) and manipulate it with the precise control (Lim and Shung, 2017). The SBAT was found to be capable of quantifying mechanical properties of various cells and molecules (Hwang et al., 2014a). Quantitative knowledge of trapping forces produced by acoustic tweezers will allow better understanding of forces associated with cellular interactions (Shao and Hochmuth, 1996). Typically, the optical trapping force was theoretically calculated based on the measured light intensity profile (Ashkin, 1992). Since the physical principle of the SBAT based on ray acoustic regime is similar to that of optical tweezers, the trapping force for the SBAT has been calculated in the same way (Lee et al., 2013). However, the measurement of acoustic intensity at frequency over 60 MHz is still not possible due to technical constraints of a hydrophone (Koukoulas et al., 2015). Therefore, it is very difficult to apply this theoretical method to an UHF SBAT. Other than the theoretical method, experimental methods including drag force (Felgner et al., 1995), power spectrum (Nørrelykke and Flyvbjerg, 2010), and equipartition theorem (Visscher and Block, 1998) have measured optical trapping forces, but not all of them are suitable for calibrating the trapping force of the SBAT. Both power spectrum and equipartition theorem methods require a very high-speed position detection system with a good image quality. However, the image quality of the SBAT is not as good as that of optical tweezers because the SBAT needs to be set above the 32 optical lens. Therefore, there are significant disadvantages to apply those two methods for the current experimental setup of the SBAT. Previous two experimental methods, namely, acceleration-mass method (Li et al., 2013) and drag force method (Lee et al., 2013) were developed to calibrated the trapping force of the SBAT, and were only used for ultrasonic frequencies less than 70 MHz and for larger particles in the range from 24 to 105 µm. This is because they have been found to suffer from large experimental error when the particle size becomes smaller. In case of the acceleration-mass method, a smooth surface relative to the particle size is considered very rough, rendering this method unpractical. External dragging force faces great challenges to keep the particle in the center stream in laminar flow as the size of particles becomes smaller. Moreover, this drag force method usually generates flow current in a larger area, but it will not satisfy the requirements of measurement in terms of precision, making the data reliable. In this chapter, I demonstrated the calibration of acoustic trapping forces on a 5 µm microparticle from the SBAT at 110 MHz using a micropipette aspiration technique (MAT). To overcome the limitations of existing method, a micropipette aspiration approach, which has been used for measuring mechanical force was investigated as a means to calibrate the trapping force of SBAT (Shao, 2002; Shao and Hochmuth, 1996). Depending on the driving conditions of the SBAT, the trapping force could range from piconewton (pN) to nanonewton (nN) (Lee et al., 2013; Lee et al., 2010a; Li et al., 2013), which is a significant advantage of the SBAT over other technologies. Previous other methodologies was limited to produce the force on the order of piconewtons only: optical tweezers (1 to 50 pN) (Kuo and Sheetz, 1992), the atomic force microscope (up to 10 pN) 33 and magnetic force apparatus (0.01 to 10 pN) (Strick et al., 1996). In this experiment, the driving condition of the SBAT was set to exert the trapping force with the nanonewton level, and the aspiration force of a micropipette could be precisely calibrated those ranges. The acoustic trapping force (F trapping ) and the trap stiffness on a 5 µm particle for a 110 MHz of the SBAT were measured against the calculated suction force from a micropipette. The trap stiffness (k), which represents the F trapping corresponding to a displacement (x) of a microparticle from the trap center, was measured. Since known suction force from a micropipette is directly applied to the calculation of acoustic trapping force, this method should be more flexible than those previously reported. Moreover, it was capable of calibrating the trapping force on only a few micrometers- particle by precisely controlling the tip size of smaller than a particle. Such a microparticle could be used as a “handle’ to mobilize cells and apply calibrated forces to cells. This work was first proposed the calibration of trapping forces produced by an UHF SBAT on a cell-sized microparticle and represents a useful and alternative tool for quantitative study of intercellular kinetics and cell fusion control (Hormeño and Arias- Gonzalez, 2006). 3.2 Experimental Procedure 3.2.1 UHF (110 MHz) Transducers Fabrication Highly press focused LiNbO 3 110 MHz single-element transducer for calibrating its trapping force was fabricated. Fabrication procedure was described in details in chapter 2. Chapter 2.4 describes how to evaluate this transducer performance. The center 34 frequency of the transducer was measured to be 110 MHz and the -6dB lateral beam was found to be 20 µm as shown in Figure 2.5 (a) and Figure 2.6 (a), respectively. 3.2.2 Micropipette Fabrication and Experimental Procedure A micropipette was fabricated with the targeting inner diameter size using a glass capillary with filament (GD-1, Narishige, NY) and a vertical micropipette puller (PC-10, Narishige, NY). Accurate suction pressures in the range from 0 to 33.3 kPa were obtained by a pressure controller (ez-gSEAL 100B, Neo biosystem, CA) and an ez-Gseal control software (NBSC Controller, Neo biosystem, CA). (a) 35 (b) Figure 3.1 Calibration of trapping forces using a micropipette aspiration technique (a) photograph (b) schematic diagram Preliminary a 5-µm particle trapping experiment using a 110 MHz transducer was already shown in Figure 2.8 (a). This experimental arrangement is similar with Figure 2.7, but adding the micropipette system at the side as shown in Figure 3.1. Two XYZ positioners (SGSP 20, Sigma KOKI Co., Japan) controlled the movement of a transducer as well as a micropipette. A bent steel needle was connected between pressure controller and micropipette at 0 degree. Trapping forces of this SBAT on a 5 µm polystyrene bead were measured under various driving conditions: peak-to-peak voltages of 4.7, 6.3 and 7.9 V and duty factors of 1%, 2%, and 3%. Pulse repetition time was fixed as a 1ms. The experimental process of trapping force calibration is described as follows. A 5 µm 36 polystyrene bead in the cell culture dish was trapped by the SBAT. The trapped microbead was moved to the micropipette tip by this SBAT, via a three axis motorized linear stage. Since the lateral beam width of this transducer is 20 µm, the bead as well as the center of the trap was moved 10 µm away from the micropipette. In that way, the micropipette was located outside of the microbeam area and would not affect the trapping performance of the SBAT. The suction pressure was started to increase from 0 Pa until the bead was completely attached to the micropipette tip as shown in Figure 3.2. (a) 37 (b) Figure 3.2 SBAT and a micropipette aspiration technique on 5-µm particle 3.2.3 Calibration of Trapping Forces and Stiffness As it is shown in Figure 3.2 (b), the bead completely stopped at a given force from a micropipette if both the trapping force on the bead from the SBAT (F trapping ) and the suction force from a micropipette (F micropipette ) equally balanced out (F trapping = F micropipette ). The suction force at the tip of micropipette could be calculated by the equation below (Shao and Hochmuth, 1996); (1) where R p is the inner radius of micropipette, ∆P is the aspiration pressure in the micropipette. Applied suction forces on the bead, calculated by the known inner radius of the micropipette (1.5-µm in this case) and the suction pressure (i.e., 0 to 33.3 kPa), were in the range from 0 to 268 nN. In order to calibrate trapping forces in a larger range, the size of micropipette should be increased. Consequently, the resolution of measurement as well as the degree of accuracy may decrease suggesting that the size of inner radius plays P R F p te micropipet D = 2 p 38 a significant role in determining both the magnitude of trapping forces and measurement requirements. Figure 3.2 (b) shows microscopic images illustrating the motion of the microbead with the SBAT and a micropipette. The white dot circle indicates the lateral ultrasonic beam. As the suction pressure increased, the trapping force balanced out with the suction force to displace the microbead away from the center of the acoustic beam. When the suction force equals with the trapping force, the microbead would stop at the position, where the trapping force was strong enough to hold the microbead. In order to generate reproducible data, the suction pressure was carefully increased with the small step size, 66 Pa, so that the microbead would be completely stopped at four designed displacements (2.5 µm, 5 µm, 7.5 µm, 10 µm). The distance between the microbead and the micropipette tip is 10 µm and less. Within such a distance, the suction force calculated above could be used to estimate the acoustic trapping force. However, flow diffusion might affect its suction force: its magnitude is well-known to be a function of the distance between the micropipette tip and bead (Shao, 2002). To improve the accuracy, the fluid dynamics module (Sánchez et al., 2008) of COMSOL Multiphysics (COMSOL Inc., Burlington, MA) was performed to determine how to compensate the decrease in force by flow diffusion before it reached the beads. Figure 3.3 presents that the entire geometry was set with the finer meshes to express more dramatic change of the pressure level. The computational configuration was built based on the experimental set-up of Figure 3.1. The inner radius and the length of the micropipette were 1.5 µm and 20 µm, respectively. The distance between the center of the bead and the tip of micropipette is 7.5 µm. Finite Element Method (FEM) solved 39 the incompressible Navier-Stokes equation to calculate the suction pressure generated by the input pressure ranged from -2.0 to -8.0 kPa at the outlet of the micropipette (the left end in Figure 3.4 (b)). Boundary conditions were defined as “normal flow and pressure” at the inlet of micropipette and “no slip” at the pipette wall. The number of elements was 108564, and the degree of freedom was 695680. The type of analysis was set as a stationary study. This model was considered converged when the estimated error in the iterative solver was below 10 -4 . If the force difference calculated by the simulation depending on the distance is defined as A, the correction factor, C, needed to compensate for the difference of forces is given by, (2) The correction factors were calculated versus the displacement (x) from the trap center at each input pressure level. The values of C via displacement at four pressure levels are displayed in Figure 3.4 (c). The corrected force, F’ micropipette , is thus, (3) The corrected force was used to calibrate the trapping force at four stop points. According to the Hooke’s law, the trapping stiffness could be calculated by the equation given below (Lee et al., 2013); (4) ( ) te micropipet te micropipet F A F C - = te micropipet te micropipet CF F = ¢ y kx F trapping + = ¢ 40 where y is the y-intercept, and x is displacement of the microbead from the trap center. When the SBAT is turned on, the microbead starts to move toward its focus. Depending on the displacement of the trapped microbead from the trap center, the trapping force changes like an elastic spring. Therefore, to determine F trapping in the ultrasonic trap, it is required to measure the trap stiffness (k), an elastic spring constant of the trap (Lee et al., 2010a). The k is calculated through the slope of linear regression by plotting F trapping – displacement from the trap center (x) curve. Therefore, trapping forces on a microbead at different displacements were calibrated. Moreover, parametric study of the trapping force was performed with varying excitation conditions including duty factors and excitation voltages. The duty factor is the fraction of the time the transducer is transmitting. Three different duty factors were used, i.e., 1%, 2%, and 3% with three different voltages, i.e., 4.7 V pp , 6.3 V pp , and 7.9 V pp . 41 (a) (b) Figure 3.3 Mesh generation and computational configuration in COMSOL 42 (a) (b) 43 (c) Figure 3.4 (a) Distribution of the normalized suction pressure on the bead surface at four different displacements from the trap center (b) Side view of computational configuration (c) Simulation of force loss over displacement from the trap center with suction pressure levels, 2.0, 4.0, 6.0 and 8.0 kPa 3.3 Result and Discussion The distribution of the normalized suction pressure on the bead surface at four different displacements from the trap center (2.5, 5, 7.5, 10-µm) is shown in Figure 3.4 (a). The x-axis of Figure 3.4 (a) graph represents the transversal arc length on the bead surface starting from the bottom pole labeled from -2.5 to 2.5-µm as displayed in the Figure 3.4 (b). In Figure 3.4 (a), the normalized suction pressure was strongest at the displacement 10 µm from the trap center and the suction pressure was observed to gradually decrease as the bead was separated from the micropipette tip. As mentioned above, the suction pressure is directly proportional to the force magnitude, and 44 consequently the force was decreased. However, since the difference of the normalized suction pressure levels at four different displacements from the trap center were shown to be less than 5%, the compensated results (F` trapping ) did not significantly affect to change. Moreover, the trend did not change with even higher input suction pressure in Figure 3.4 (c). The trapping force is proportional to the displacement of the microbead from the center of acoustic beam as an elastic spring as shown Figure 3.5. All data are expressed as means ± standard deviation of five measurements. The SBAT with higher excitation voltages or duty factors exerts to stronger trapping forces and consequently, stronger suction forces were needed to displace the microbead at the same position. With the excitation voltage of 4.7 V PP , a suction force of 22.7 ± 4.4 nN was applied to displace the microbead 10 µm away from the trap center; however, when the excitation voltage was increased to 7.9 V PP , a stronger force of 40.7 ±7.4 nN was required to generate the same displacement of the microbead. The calculated results could be used to accurately calibrate the trapping force, which make the SBAT works as a non-contact force- measuring tool. 45 (a) 46 (b) Figure 3.5 Relation between trapping force and displacement from the trap center for (a) different values of input voltage with PRF = 1kHz and DTF = 1% (n=5) (b) different values of DTF with PRF =1kHz, and a constant input voltage = 6.3 V PP (n=5). For the parametric study of the SBAT, Figure 3.5 (a) presents the relation of the F trapping and the displacement for varying excitation voltages with the pulse repetition frequency (PRF) of 1kHz and the duty factor of 1%. The stiffness values were 1.4, 1.6, 2.9 nN/µm for the excitation voltages of 4.7, 6.3, and 7.9 V pp , respectively. It indicates that higher excitation voltage results in the stronger trap stiffness. Similar tendencies are 47 shown in Figure 3.5 (b). As the duty factor increased from 1% to 3%, the trap stiffness was also increased from 1.7 to 4.7 nN/µm with the PRF of 1 kHz and the excitation voltage of 6.3 V PP . In summary, the stronger trapping force and stiffer acoustic trap for a given x were implemented if the input voltage and the percentage of duty factor were increased. In previous study, stronger trapping forces at the low frequency of 30 MHz were induced by higher acoustic pressures (Lee et al., 2010a). Even though the measurement of acoustic pressure at frequency above 60 MHz is not available, it is proved that acoustic pressure generated from the transducer is usually proportional to the input voltage to the transducer (Johns et al., 2007), and the acoustic pressure exerted at the focus is also positive related to the duty factor (Hwang et al., 2014b). Therefore, higher input voltages and higher duty factors may lead to higher acoustic pressures, which suggests this measurement results are in an agreement with that previously measured in lower frequency range (Lee et al., 2010a). Moreover, nanonewton trapping forces estimated in this study appeared to be reasonable as similar levels of trapping forces obtained by Lee both theoretically and experimentally, albeit at low frequencies (Lee et al., 2013; Lee et al., 2010a). Since the trapping force of acoustic tweezers varies depending on a center frequency, an aperture size and an f-number, its magnitude also may vary from piconewton to nanonewton. The data obtained in this study can be better validated if the measurement of acoustic intensity generated by the transducer at UHF is made possible. Accurate measurement of the trapping force and the precise control of the microbead were achieved by producing the small inner radius of the micropipette tip (1.5 48 µm). It is shown that the proposed method, MAT could be used to calibrate the trapping force of the UHF SBAT on the 5 µm polystyrene bead. Comparing with previous drag force methods and acceleration methods, the measurement error was significantly reduced when the size of the manipulated object was reduced to such a level. In biological experiment, applying known forces to manipulate the object sized 5 µm or less is a necessary condition to investigate the intercellular adhesion process and the mechanical properties of a cell membrane (Hwang et al., 2014a; Lam et al., 2012). The SBAT have minimal effects on cellular properties due to its characteristics of a non- contact and a non-label manipulation. In addition, the proposed calibration method will provide quantitative information of the acoustic trapping force. The micropipette cross-section in this experimental approach plays a key role in affecting the accuracy of the measurement. It controls the minimum aspiration force as well as the step size of the force increase at current limits of pressure control capability. Consequently, it manages the trapping force measurement range and precision, which determine if the experimental arrangement meets the requirement for a particular cellular study. The cross-section of the micropipette tip used in this study should be smaller than a single cell with the measurement range from a few nNs to a few hundred nNs. As a result, nanonewton forces at cellular-adhesion sites and the correlated intercellular signaling response of mechanotransduction could be characterized with the calibrated trapping force. Smaller trapping forces in order of a few hundred piconewtons may be measured with an upgrade of current micropipette aspiration device and is presented in the next chapter for measuring inter RBC forces. 49 3.4 Concluding Remarks In this chapter, transverse two-dimensional trapping forces from the 110 MHz LiNbO 3 focused transducer were calibrated using a MAT. Even though various types of transducers for the SBAT at frequency above 60 MHz were fabricated in past few years, trapping forces produced by those transducers have never been calibrated. The results reported in this study demonstrate that the proposed method could be applied to calibrate the trapping force on the cell-sized object. Moreover, this technique was applied to calibrating a transducer at even higher frequency, allowing trapping even smaller particle in the next chapter. The measured trapping force was in the range of nanonewton and the trap stiffness was in the range of nanonewton per micrometer. An investigation into transducer excitation parameters indicates that a higher excitation voltage and a larger duty factor yield a stronger force and a stiffer trap. In the next chapter, the calibrated acoustic trapping device will be used to measure intercellular adhesion forces during RBC aggregation. 50 CHAPTER 4 Quantification of Inter-Erythrocytes Forces with UHF SBAT *In this chapter, a substantial portion of the original paper including figure 4.1~4.8 was reprinted with permission from H. G. Lim and K. K. Shung, “Quantification of inter- erythrocyte forces with ultra-high frequency (410 MHz) single beam acoustic tweezer”, Annals of Biomedical Engineering, vol. 45, no. 9, pp. 2174-2183, 2017 (© [2017] Springer). 4.1 Background RBC aggregation is well-established factor responsible for the flow properties of blood. It affects blood circulation and tissue perfusion including microcirculatory blood flow dynamics and venous flow resistance. Various circulatory disorders such as malaria, sickle cell disease (Tripette et al., 2009), and system lupus erythematosus (SLE) (Reid and De Ceulaer, 1999) were led by abnormal RBC aggregation. RBC agglutination is caused by intermolecular attractive forces such as electrostatic forces, Van der Wall force, and hydrogen bonds. In previous studies, two different models have been proposed to explain RBC aggregation: the Cross-Bridge model and Depletion model. The Bridge model proposed that fibrinogen in plasma and the macromolecules non-specifically absorb onto the RBC membrane and form bridges between adjacent cells (Chien and Jan, 1973; Chien and Sung, 1987). Fibrinogen mediated aggregation of RBCs could induce reversible rouleaux formation, and its aggregation was directly proportional with the concentration of fibrinogen (Chien et al., 1983; Marton et al., 2001). The depletion theory 51 proposed that the surrounding macromolecules around RBCs were expelled from the region between RBCs, and the concentration of macromolecules depleted in this region compared to the bulk phase (Asakura and Osawa, 1954). This osmotic pressure difference leaded to solvent displacement in to the bulk phase, resulting in an attraction among RBCs (Neu and Meiselman, 2002; Steffen et al., 2013). The quantitative knowledge of inter RBC forces allows a better understanding of the mechanism of RBC aggregation. In the last few decades, the mechanics of RBC interaction at a single cell level have been studied by different technologies: scanning electron microscopy (SEM) (Jan and Chien, 1973), micropipette aspiration technique (MAT) (Buxbaum et al., 1982), atomic force microscopy (AFM) (Steffen et al., 2013), and optical tweezer (OT) (Bronkhorst et al., 1997). The first estimation of interaction energy density between RBCs in autologous plasma measured using a MAT was about a few µJ/m 2 (Buxbaum et al., 1982), and the AFM allowed to measure the depletion induced adhesive force between two RBCs which was found to be in the range from 14 to 170 pN (Steffen et al., 2013). However, direct contact with objects was required to measure the force for both methods, causing the physical damages on their surface of RBCs. More recently, many aspects of RBC behaviors have been studied in optical tweezers, which is a non-contact tool as well. Maklygin et al. fabricated double beam optical tweezers to measure interactive forces between RBCs in a aggregate (Maklygin et al., 2012). Khokhlova et al. measured aggregation forces and aggregation velocities in RBC pairs obtained for healthy and SLE blood samples using double-beam optical tweezers (Khokhlova et al., 2012). Moreover, Lee et al. compared between RBC 52 aggregating and disaggregating forces in both autologous plasma (Lee et al., 2016a) and in protein solutions (Lee et al., 2016c) to study dynamic RBC interactions depending on its environment conditions. Aggregation forces measured by optical tweezers were found to be the range only from 8.4 to 30 pN (Khokhlova et al., 2012; Lee et al., 2016b; Lee et al., 2016c; Maklygin et al., 2012). However, in cases where RBCs interacted with stronger forces than the maximal force of optical traps limited on the order of dozens of pN, the optical trap force could no longer induce for further disaggregation, hence making it impossible to measure those forces. In approximately 90% of the experiments, the RBCs were either connected by small tethers or still in contact with each other, meaning that they were not totally separated. (Khokhlova et al., 2012; Khokhlova et al., 2010). Moreover, as additional limitation of the optical tweezer, the laser sources easily introduced thermal effects, resulting in biological damages to the sensitive surface area of RBCs, and eventually the force of surface affinities between two RBCs was not reliable and constant. As an alternative tool for measuring inter RBC forces, the SBAT may have the following advantages over optical tweezers: 1. Stronger trapping forces in ranges from pN to nN depending on the driving conditions (Lee et al., 2013; Lee et al., 2010a; Li et al., 2013; Lim et al., 2016), 2. Non-mechanical contact and non-label tweezer tool leading less damage on objects 3. More suitable tool for vivo experiment with a larger depth of penetration in opaque medium such as blood. For these reasons, to date, the SBAT have been developed to study the mechanical property of the cancer cell membrane (Hwang et al., 2014a) and intracellular calcium signaling properties of cancer cells (Hwang et al., 53 2015). Previous studies were carried out with transducers at frequencies under 200 MHz, which produced a spatial resolution in the range from tens to hundreds of micrometers. This is because the fabrication of an UHF transducer is challenging and time consuming process requiring the precise control of fabrication parameters of the UHF transducer. In this chapter, I designed and fabricated 410 MHz ultrasonic transducers, which had a beam width of only a few micrometers beam width, thus it allows the precise control of a microparticle, a nanoparticle, or a single cell. In addition, a calibration system based on MAT for trapping force of the SBAT at UHF was developed in chapter 3. A 410 MHz transducer was used to trap and detach RBC from a RBC aggregate. Hence, I measured the intercellular adhesion forces between two RBCs against the pre-calibrated trapping force using a MAT. The SBAT at UHF with an ultrasonic beam width smaller than diameter of the RBC is a key advantage over previous SBATs. This small beam width allowed trapping a single RBC preventing any significant disturbance to the adjacent RBCs. The study of enhancement of RBC aggregation has been of interest in the field of physiology and biomedicine for patients suffering from blood disorders. Using this new method, SBAT of development in measuring inter-force between RBC would be potentially used in clinical diagnosis. 4.2 Experimental Procedure 4.2.1 UHF (410 MHz) Transducers Fabrication Highly press focused LiNbO 3 410 MHz single-element transducer for quantifying inter RBC forces was fabricated. Fabrication procedure was described in details in 54 chapter 2. LiNbO 3 was lapped down to its designed thickness of 7.1 µm with an extreme care, and diced into the size of 250 µm. Chapter 2.4 describes how to evaluate this transducer performance. The center frequency of the transducer measured to be 410 MHz and the -6dB lateral beam was found to be 6.5 µm as shown in Figure 2.5 (c) and Figure 2.6 (b), respectively. 4.2.2 Blood Sample Preparation Fresh blood was obtained from ten healthy donors by a fingertip needle prick. RBCs were washed three times with isotonic phosphate buffered solution (PBS) by centrifuging at 3000 g for 5 min, and the separated plasma was stored for later re- suspension. 1-µL of RBCs was diluted in 100 uL PBS. Microspheres of 3.78 µm diameter (Bangs Laboratories Fishers, IN) were centrifuged in distilled water at 3000 g for 5 min. Three times washed microspheres were re-suspended in 0.1% poly-lysine solution (Sigma-Aldrich, St Louis, MO) and stored at room temperature for 4 hours for coating. The coated microspheres were washed one time and diluted to 2.5 x 10 4 /µL in PBS. 100- µL poly-lysine coated microsphere suspension was mixed with 100 µL diluted RBCs suspension so that the ratio of RBCs and beads was 2:1, and waited for 1 hour resulting in strong adhesive interaction between RBCs and coated microspheres (Lam et al., 2012). After that, RBCs and microspheres were suspended in 1 mL autologous plasma for cell aggregation. The resultant solution was added into a petri dish with an acoustically transparent mylar film as the bottom surface. The poly-lysine coated bead made a strong attachment with mylar film as well as RBCs aggregation. Subsequently, 0.1% serum 55 bovine albumin (BSA) solution was added to the suspension in order to prevent RBCs from sticking to the mylar film. 4.2.3 RBC Experiments The experimental arrangement for this 410 MHz SBAT is shown in Figure 4.1. Sinusoidal burst signals at 410 MHz, generated by a function generator (Stanford Research Systems, Sunnyvale, CA) and amplified by a 50-dB power amplifier (525LA, ENI, Rochester, USA) were used to drive the transducer. The motion of RBCs was observed using an inverted microscope (Olympus IX-71, Center Valley, PA) and recorded via a CMOS camera (ORCA-Flash2.8, Hamamatsu, Japan). How a single RBC can be manipulated by SBAT is shown in Figure 2.7 . A single RBC could be trapped by the SBAT at 410 MHz with a duty factor of 1%, a pulse repetition frequency (PRF) of 1 kHz, and a peak-to-peak input voltage (V pp ) of 7.94. The black arrow in Figure 2.7 (1-4) shows a single RBC movement along the SBAT direction. 56 Figure 4.1 Single RBC trapping with SBAT. Schematic of experimental systems. RBC was trapped by a 410 MHz transducer driven a duty factor of 1%, a PRF of 1 kHz, and a peak-to-peak input voltage 7.94 of V pp . Figure 4.2 and 4.3 illustrates experimental steps of measuring inter RBC forces. The poly-lysine coated bead attached to the bottom of the petri dish and RBCs aggregation due to the poly-lysine coating. Two RBCs (labeled as RBC-1 and RBC-2 in Figure 4.2 and 4.3) were aggregated by autologous plasma. The focal point of the trap center set up to be located 10 µm apart from the center of the RBC-2 as shown in Figure 4.3 (a) via a three axis motorized linear stage (SGSP 20, Sigma KOKI Co., Japan). The transducer was turned off at first and turned on with an input voltage starting from 0 V pp 57 with a duty factor of 1%, a PRF of 1-kHz. As the 410 MHz transducer increased the input voltage with a step size of 0.31 V pp , the RBC-2 gradually towed into the acoustic trap with deforming as shown in Figure 4.3 (b-e). The input voltage was increased until the stronger trapping force induced the disaggregation of two RBCs as shown Figure 4.3 (f). The distance between the center of the RBC-2 and the trap center when the disaggregation employed were recorded using a camera and analyzed by an image analysis software (Metamorph, Molecular device, Sunnyvale, CA). Figure 4.2 Schematic steps of measuring the inter RBC force. 58 Figure 4.3 Measurement of the inter RBC forces. (a) A poly-lysine coated bead was attached to the bottom of the dish as well as a RBC aggregate. The transducer was fixed at one position and input voltage was increased starting from 0 V pp . (b-d) As the transducer gradually increased the input voltage, the RBC-2 is pulled into the acoustic trap by deforming. (e) at input voltage 7.94 V pp , the RBC-2 is completely detached from the RBC-1. (f) microscopic images of disaggregated RBCs. Red circles, yellow solid circles, and blue solid circles represent the acoustic trap zone, the center of acoustic trap and the center of RBC-2 respectfully. n=10 4.2.4 Trapping Force Calibration using a MAT Calibration of the trapping force from the SBAT was made with an upgraded micropipette aspiration technique (MAT) described in chapter 3. The main advantage of this technique is its capability of measuring the trapping force ranging from pN to nN depending on transducer’s driving conditions. Fabrication of micropipette devices was same as that of chapter 3.2.2, but the targeting inner diameter size was 1.5 µm. A pressure controller was used to accurately generate the suction pressure in a range from 0 to 33.3 kPa, controlled by an ez-Gseal control software. As shown in Figure 4.4 (a), the schematic diagram of the experimental arrangement, the SBAT and the micropipette controlled by two XYZ positioners, respectively, were used to manipulate the RBC in a 5 µm a b c d e f RBC-1 Bead RBC-2 5 µm 5 µm 5 µm 5 µm 5 µm i ii iii RBC-1 RBC-2 Bead 59 petri dish. A bent steel connector was connected the pressure controller with the micropipette lining at 0 degree. Figure 4.4 Calibration acoustic trapping force using a MAT. Schematic of the experimental arrangement. The RBC was trapped by a 410 MHz transducer driven at a duty factor of 1%, a PRF of 1kHz, and a peak-to-peak input voltage of 7.94 V pp . The micropipette was set up at the side of the RBC and the suction force was increased starting from 0. The RBC is moved to the tip of micropipette as the two forces, the suction force and the trapping force are balanced out. The procedure of the calibration experiment is shown in Figure 4.4 (b). Figure 4.5 shows microscopic images illustrating the change of RBC motion during calibration. A randomly selected RBC in the dish was trapped and located at 10 µm away from the micropipette tip by the SBAT so that the trapping action of the SBAT was not affected by 60 the presence of the micropipette. Red dot circles in Figure 4.5 indicate the ultrasonic trap at focus. The trapping forces of the SBAT acting on the RBC were under the same driving conditions of the SBAT as those of the inter RBC force experiment: a duty factor of 1%, a PRF of 1-kHz and the peak-to-peak voltages 7.94 V pp . The suction pressure was increased from 0 Pa at the step size of 2.6 Pa until the RBC was completely attached to the tip of the micropipette as shown in Figure 4.5 (e). As the suction pressure was increased, the acoustic trapping force was equally balanced out by the suction force of the micropipette to displace the RBC from the center of acoustic beam to the micropipette tip. Suction forces were recorded when the RBC was located at four designated displacements from the trap center (1.0 µm, 2.0 µm, 3.0 µm and 4.0 µm). It is worthwhile to note that as the suction pressure exceeded a certain level, RBC was completely sucked into the micropipette. Figure 4.5 Microscopic images of calibrating trapping force. Red circles represent the acoustic trap zone. (a) the RBC was trapped by the transducer and located 10 µm from 61 the micropipette tip. (b-e) The suction force from the micropipette was turned on and was increased until the RBC was attached to the tip. (f) The RBC was completely attached to the micropipette tip and started to deform. (g) The RBC was completely sucked into micropipette and disappeared. 4.2.5 RBC Viability Test A viability assay was carried out with Calcein AM, (Invitrogen Corp., Grand Island, NY) after the cell trapping experiment. Calcein-AM is a vital dye that rapidly enters live- cells converting to green fluorescent. To assess the cell viability, Calcein-AM was prepared as a stock solution of 1 mM in dimethylsulfoxide stored at room temperature. At a final concentration of 10 µM of Calcein-AM it was added into the sample chamber containing the RBC. Fluorescence imaging of the RBC was obtained by a microscope with an excitation of 488 nm and an emission of 532 nm. The intensity of green fluorescence was normalized before (control) and after trapping (10, 20 and 30 min), and statistical analysis including mean and standard deviation with the sample size 10 were calculated. After all, the two–tail paired t-test with a p-value < 0.05 was examined to check their significance level of difference. 4.3 Experimental Results Blue closed circles and red circles in Figure 4.3 represent the center of the RBC-2 and the trap area, respectively. Trap center was originally located 10 µm from the blue closed circle in Figure 4.3 (a). As the input voltage of the transducer was increased from 0 V pp while other parameters were fixed i.e., a duty factor of 1% and a PRF of 1-kHz, the RBC-2 was gradually moved closer to the focal zone. When two RBCs started to be disaggregated as shown in Figure 4 (e) and the distance between the RBC-2 and the trap 62 and the applied voltage were found to be 3.76 ± 0.84 µm and 7.94 ± 2.35 V pp, respectively. The acoustic trapping force to disaggregate two RBCs equal to the inter RBC force (Trapping force = inter RBC force). In order to find the trapping force at the displacement, 3.76 µm from the trap center, a calibration experiment was performed. F trapping calculation and its principles were detailed explanation was described in chapter 3.2.3. F trapping could be calculated from the equation of the suction force at the micropipette tip below (Shao and Hochmuth, 1996); F micropipette = πR 2 3 ∆P (1) where R p is the inner radius of a micropipette, ∆P is the aspiration pressure in a micropipette. The force generated from a micropipette (F micropipette ) was equally balanced out by the acoustic trapping force (F trapping ). The force measurement range of the pressure controller was from 0 to 6.54 nN based on the inner radius of the micropipette (0.25-µm in this study) and the suction pressure (i.e., 0 to 33.3 kPa). According to the Hooke’s law, the trapping stiffness could be calculated by the equation given below (Lee et al., 2013); F trapping = kx + y (2) where y is the y-intercept, and x is the displacement of RBC from the trap center. An UHF SBAT generates a highly focused acoustic trap to induce the RBC toward its trap center. Depending on the displacement of the trapped RBC from the trap center, the trapping force changes like an elastic spring. The trap stiffness (k), an elastic spring constant of the trap (Lee et al., 2010a) provides the F trapping in the ultrasonic trap zone. 63 The k was calculated to be 102.9 pN/µm through the slope of linear regression by plotting F trapping – displacement from the trap center curve. Therefore, the trapping forces on the RBC could be calibrated a function of the displacements in Figure 4.6. Since the final displacement was measured as 3.76 µm between the RBC-2 and the trap center, the final trapping force was estimated to be 391.0 ± 86.4 pN from the graph shown in Figure 4.6. Figure 4.7 shows the RBC’s viability before (control) and after (10, 20, and 30 min). After trapping force applied to the RBC, fluorescence intensity of Calcein AM was measured in Figure 4.7 (a). All of the intensity was normalized in Figure 4.7 (b) and compared with the control group using two tailed t- test with a p-value < 0.05. No significant difference between them was found as the p-value was 0.20 which was greater than 0.05. As a result, I may conclude that the trapping force produced little effect on the RBC viability under the given acoustic driving conditions. 64 Figure 4.6 Calibration graph for the 410 MHz transducer driven at a duty factor of 1%, a PRF of 1 kHz, and a peak-to-peak input voltage of 7.94 V pp showing the relation between the trapping force and the displacement from the trap center and the RBC center. n=10 65 (a) (b) Figure 4.7 Trapping force effects on RBC viability. (a) Fluorescence image before trapping (control) and after trapping (10, 20, 30 min). (b) Normalized cell viability. The fluorescence intensity was normalized and compared with the control group. n=10 4.4 Discussion This study experimentally demonstrated that the capability of a SBAT at UHF for measuring inter RBC forces by pulling one of the RBCs from the RBC aggregate. Previous studied conducted several cellular SBAT applications. The SBAT was used to study the mechanical properties of human breast cancer cells including MCF-7 (Hwang et al., 2014a), SKBR-3 (Hwang et al., 2015), and MDA-MB-231 (Hwang et al., 2016). Also, 0.8$ 0.85$ 0.9$ 0.95$ 1$ control' 10'min' 20'min' 30'min' Normalized'Cell' Viability'' control 10 min 20 min 30 min a b 5 µm 5 µm 5 µm 5 µm 0.8$ 0.85$ 0.9$ 0.95$ 1$ 1.05$ 1.1$ control$ 10$min$ 20$min$ 30$min$ Normalized+ Cell+Viability++ 66 the SBAT was shown to be capable of manipulating cell-sized microparticles inside of an excised blood vessel (Li et al., 2014). Although previous versions of the SBAT’s capability of measuring cell deformability was previously demonstrated (Hwang et al 2016), it was neither possible to obtain absolute values of elastic constants of cells including RBC nor possible to measure the interactive forces between cells due to the frequency of the SBAT. This is because the beam width of an ultrasonic transducer is inversely proportional to the center frequency. If the beam size is larger than the dimension of a RBC, the radiation force generated by the transducer might disturb the adjacent RBCs as well making the interactive force measurements unreliable. Therefore, fabricating UHF transducer, which can generate acoustic beam size less than the dimension of a RBC (~7.5 µm), was a key to allow this experiment to be successfully carried out. The 410 MHz ultrasonic transducer with beam width, 6.5 µm fabricated for this work allows the trapping of a RBC without producing disturbance to adjacent RBCs as shown in Figure 4.8. Fabrication of the 410 MHz transducer was challenging because the piezo-electrical material had to be lapped down to the targeted thickness, 7.1 µm, with an extreme care. In addition, the extremely small thickness of the piezoelectric material makes it very easy to be broken during the mechanical pressing focus using a heated bearing ball. 67 Figure 4.8 Smaller size of ultrasonic bead than the diameter of a RBC preventing any disturbance to adjacent RBCs. The relative deformability of cancer cells was measured by previous SBAT at 200 MHz, but no quantitative absolute estimate of any cellular elastic constants could be obtained (Hwang et al., 2014a; Hwang et al., 2015). One of the reasons is that the measurement of the acoustic trapping force at frequency over 60 MHz is not possible due to the technical constraints of hydrophone and system (Koukoulas et al., 2015). However, I successfully calibrated the trapping force of 110 MHz transducers using a micropipette aspiration technique. By applying the MAT with a reduced inner diameter of the micropipette, the trapping force of the 410 MHz transducer was found to be in the order of a few hundred piconewtons. As shown in Figure 4.6, the calibrated trapping forces ranged from 100 to 500 pN and the trap stiffness, k calculated to be 102.9 pN/µm. 7.5$µm$ 410$MHz$ transducer RBC 68 Consequently, the inter RBC force was found to be 391 pN by interpolating from the pre- calibrated trapping force. Optical tweezers measured the RBC aggregation and disaggregation forces in the range of a few pNs (Khokhlova et al., 2012; Lee et al., 2016b; Lee et al., 2016c; Maklygin et al., 2012). However, due to the limitation on the maximal trapping force produced by the optical tweezers, measurement could not be made if RBCs aggregation forces were stronger than the optical trapping force. Since they consider only the cases where successful break-ups of an aggregate were achieved in their experiments, aggregation forces measured by optical tweezers might be underestimated. This may be the primary reason that the aggregation force, measured by the SBAT is much stronger than that estimated by optical tweezers. Since the SBAT has the advantage of being capable of generating larger trapping force from pN to nN depending its driving conditions, this method may be a viable alternative to optical tweezers. 4.5 Concluding Remarks In this chapter, the inter RBC force at a single cell level was directly measured based on the pre calibrated trapping force produced by a 410 MHz LiNbO 3 focused transducer. Since the focal beam diameter of the 410 MHz ultrasonic transducer used in this SBAT was only 6.5 µm, which was smaller than that of a RBC ~7.5 µm, it was made possible to directly apply the beam to a single RBC without any disturbance to the neighboring RBC during the trapping experiments. The trapping forces produced by this UHF SBAT can be quantitatively estimated with a micropipette aspiration technique. 69 Based on pre-calibrated acoustic trapping forces, the magnitude of inter RBC forces was found to be 391.0 ± 86.4 pN. In the near future, efforts will be made to use this technology to assess the interactive forces between other types of cells including the adhesive forces between white blood cells and the endothelial cells of the blood vessel surface. 70 CHAPTER 5 Investigation of Cell Mechanics using SBAT 5.1 Background The mechanical properties of biomaterials, tissues, and cells have gained attention for their tissue response and the cellular function and regulation (Deguchi and Sato, 2009; Titushkin and Cho, 2009). The cytoskeleton, the most significant cellular mechanical component, can mediate cell response by changing its biomechanical environment such as cell shape, cell deformation, and external pressure (Janmey and McCulloch, 2007). Recently, it was discovered that various blood disorders and infectious diseases disrupt RBC morphology and biomechanical characteristics (Diez-Silva et al., 2010). Moreover, the cancer cell mechanics, the mechanical stiffness level indicated the determination of the invasion potential of the cancer cell (Swaminathan et al., 2011). Therefore, investigating cell mechanics could play a key role in predicting and evaluating such diseases in medical and clinical fields. Currently available methods for measurement of cell mechanics include atomic force microscope (AFM), optical tweezer (OT), magnetic tweezer, and stretchable substrates(Bao and Suresh, 2003); however, there are some drawbacks to directly applying those methods to cellular-level studies. An AFM consists of a cantilever with the probes to make direct contact with the surface of a cell for force spectroscopy measurements, which can cause the destruction of the cell membrane (Rico et al., 2005). The OT is a well-established non-contact tool for manipulating the cells and nano/microparticles with a focused laser beam. OTs were used for investigating RBC 71 elasticity (Hénon et al., 1999) and aggregation force (Lee et al., 2016b; Lee et al., 2016c) by pulling a microbead attached to RBCs across the cell surface. However, because the force an optical trap can generate is limited to an order of piconewton level, for such cases that require measurement of stronger forces than maximal optical trapping force, it is not possible to examine their properties using OTs. The magnetic tweezer is an experimental manipulation technique using magnetic beads to measure cellular forces and the local viscoelasticity in living cells (Bausch et al., 1999; Walter et al., 2006). One of its shortcomings is that the magnetic beads need to be loaded inside of a cell (Bausch et al., 1999). In order to investigate more reliable way to estimate of a cell’s mechanical properties, an alternative tool needs to be developed that is a non-invasive to the cell surface and has the capability to produce stronger forces. The single-beam acoustic tweezer (SBAT) is an advanced non-contact tool that can generate a trapping force in the range from piconewton to nanonewton (Li et al., 2013; Lim et al., 2016; Lim and Shung, 2017) and a radiation pressure of several megapascals (MPa) (Hwang et al., 2016; Lee et al., 2010a). For this reason, precise cell manipulation under the SBAT have been proposed and used for studying cellular activities (Hwang et al., 2015; Lam et al., 2016). Recently, a SBAT with a 200 MHz ultrasonic transducer measured the elastic properties of cultured breast cancer cells (Hwang et al., 2014a). The cell membrane was stretched by a microbead attached to a target cell with an acoustic trap resulting in local membrane deformation. More recently, the deformability of various suspended breast cancer cells was quantitatively measured under the SBAT, so that the different degrees of cancer cell 72 invasiveness were compared (Hwang et al., 2016). This experiment investigated cells in the suspension state without any labeling or material attached, which allows for more practical applications for biomedicine. However, the researchers could not provide the absolute value of Young’s modulus because it was only possible to compare invasiveness levels based on deformability. Hence, it was desirable to develop the SBAT for the absolute quantification of the mechanical properties of cells. In this study, I demonstrated the ability to use SBAT to quantitate the mechanical properties of an unlabeled suspended cell using a 45 MHz transducer. As mentioned above, previous studies were not able to provide the quantitative elastic modulus because they could not determine standardized reference data of cell deformability (Hwang et al., 2016). As an extension of previous work, in the present experiment I utilized agarose hydrogel spheres, cell-mimicking phantoms that exhibited various mechanical properties, to calibrate the deformability of cells. In these days, agarose hydrogel is widely used as tissue-engineered cartilage and stiff biomaterial (Zhou et al., 2016), and its mechanical properties has successfully quantified by ultrasound elastography as a nondestructive method (Chung et al., 2015). Selected agarose hydrogel spheres possessed similar mechanical properties to native cells. A single suspended cell or a hydrogel sphere was trapped by the SBAT and its deformability was examined under the transducer’s various driving conditions. After a deformability experiment using the SBAT, hydrogel spheres were mechanically tested using a micropipette aspiration technique (MAT) to determine their Young’s modulus, which functioned as a reference data. Due to its simplicity, a MAT has been used for investigating the mechanical properties of a cell and a cell-sized 73 microsphere (Lee and Liu, 2014). With the deformability relationship between cells and spheres in addition to the Young’s modulus of the hydrogel spheres in 0.1~1.2% concentration, the indirect quantitative measurements of cell elasticity was assessed. Furthermore, mechanical properties of breast cancer cells with different degrees of invasiveness were quantitatively compared. Therefore, those experimental results revealed the feasibility of non-contact evaluation of cell mechanics with label-free using the SBAT. 5.2 Experimental Procedure 5.2.1 High Frequency (45 MHz) Transducer Fabrication For this study, the SBAT pushed the whole part of a cell to deform, so the focal area should be larger than a cell-size. Therefore, the targeting frequency was 45 MHz because its wavelength is approximately 30 µm, close to the breast cancer cell size. The high-frequency lithium niobate (LiNbO 3 ) press focused transducer was fabricated following procedure I used previously (Lim et al., 2016). Since 36º rotated Y-cut LiNbO 3 single crystal offers a high electromechanical coupling coefficient (k t ~49%) and a low dielectric permittivity (ε s ~ 39), the transducer can be designed to form a large aperture size of the material and offers a high sensitivity. Detailed procedure of this fabrication is as follows: 1. The software, a Krimholtz, Leedom, and Matthaei model (PiezoCAD, Sonic Concepts, Bothell, WA) was used to design an optimal aperture size and a thickness of LiNbO 3 , matching, and backing layer. LiNbO 3 was manually lapped down to 66 74 µm. Chrome and gold electrodes (Cr/Au, Nano-Master, TX, USA) were sputtered with a thickness of 1500 Å on front side of the material. 2. The first matching layer, a mixture of 2-3 µm silver particles (Silver; Aldrich Chemical Co., MO, USA) and Insulcast 501 epoxy (Insulcast 501, American Safety Technologies, PA, USA) was attached to the front side of LiNbO 3 and was lapped down to 10 µm. Chrome and gold electrodes were sputtered on backside of LiNbO 3. Conductive epoxy (E-solder 3022, Von Roll Isola, USA) with a thickness of 1 mm was attached to the backside of the material. 3. The acoustic stack (LiNbO 3 , matching layer, and backing layer) was turned down to the diameter of 3 mm and then was concentrically placed in the brass housing. The gap between the acoustic stack and the brass was filled with an insulating epoxy (Epo-tek 201, Epoxy Technologies, Billerica, MA) in order to prevent the electrical short. 4. Mechanical press focusing on the surface of LiNbO 3 was performed using a heated bearing ball to obtain an f-number 1.5 (the focal distance of 5 mm and the aperture diameter of 3.3 mm). The press focused surface of LiNbO 3 was sputtered again by chrome and gold electrodes with a thickness of 1500 Å. 5. The single lead wire was connected to the backing layer, and then the SMA electrical connector was built into brass housing connecting the single lead wire. The second matching layer, a parylene film (12 µm) was coated the outside of the transducer using PDS 2010 Labcoater (SCS, Indianapolis, IN, USA). 75 5.2.2 Transducer Performance To investigate the performance of the fabricated transducer, JSR (Pittsford, NY, USA) model, DPR 500 pulser/receiver was connected to the transducer and exited the electrical impulses at 500 Hz repetition rate at 50 damping. A pulse-echo response and a frequency spectrum were measured as shown in Fig. 1. The center frequency was found to be 45 MHz, and the -6 dB fractional bandwidth was 75%. Two-dimensional lateral and axial intensity of spatial peak temporal average (I SPTA ) was measured using a needle hydrophone (Precision Acoustics, UK) as shown in Fig. 2. The driving condition was as follows: frequency of 45 MHz, input peak to peak voltage of 20 V, cycle numbers of 10, and pulse repetition frequency (PRF) of 500 Hz. The -3 dB lateral beam width was measured to be 60 µm. The beam profile and ISPTA was observed to be symmetric at the focal point in lateral axis. 76 (a) (b) 0 20 40 60 80 −15 −10 −5 0 Frequency (MHz) Amplitude (dB) 4.9 5 5.1 5.2 5.3 −0.4 −0.2 0 0.2 0.4 Amplitude (V) Distance (mm) (a) (b) (c) 0 20 40 60 80 −15 −10 −5 0 Frequency (MHz) Amplitude (dB) 4.9 5 5.1 5.2 5.3 −0.4 −0.2 0 0.2 0.4 Amplitude (V) Distance (mm) (a) (b) (c) 77 (c) Figure 5.1 Fabrication of a 45 MHz transducer. (a) Receive-echo response. (b) Frequency spectrum. (c) Photograph of a 45 MHz transducer (a) 0 20 40 60 80 −15 −10 −5 0 Frequency (MHz) Amplitude (dB) 4.9 5 5.1 5.2 5.3 −0.4 −0.2 0 0.2 0.4 Amplitude (V) Distance (mm) (a) (b) (c) (b) !150% !100% !50% 0% 50% 100% 150% 0% 10% 20% 30% 40% 50% 60% !150% !100% !50% 0% 50% 100% 150% Ispta&(mW/cm 2 )& X/axis&(µm)& 50!60% 40!50% 30!40% 20!30% 10!20% 0!10% Y/axis&(μm)& 0% 10% 20% 30% 40% 50% 60% !120% !90% !60% !30% 0% 30% 60% 90% 120% Ispta&(mW/cm 2 )& Transverse&distance&from&focus&(µm)& (a) 78 (b) Figure 5.2 Characteristics of a 45 MHz transducer. (a) Lateral beam profile. (b) 2- dimensional I SPTA profile. 5.2.3 Cell Preparation Human breast cancer cell lines: MDA-MB-231, MCF-7, and SKBR-3 were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco’s modified eagle medium (DMEM). Phosphate Buffer Solution (PBS) was purchased from Invitrogen (Grand Island, NY) for maintaining the cells during the experiment. A trypsin- EDTA solution obtained from Invitrogen (Grand Island, NY) was used for detaching the cells from the bottom of the petri dish to be in a suspension state. (b) !150% !100% !50% 0% 50% 100% 150% 0% 10% 20% 30% 40% 50% 60% !150% !100% !50% 0% 50% 100% 150% Ispta&(mW/cm 2 )& X/axis&(µm)& 50!60% 40!50% 30!40% 20!30% 10!20% 0!10% Y/axis&(μm)& 0% 10% 20% 30% 40% 50% 60% !120% !90% !60% !30% 0% 30% 60% 90% 120% Ispta&(mW/cm 2 )& Transverse&distance&from&focus&(µm)& (a) 79 5.2.4 Cell and Sphere Deformation by SBAT Agrarose hydrogel spheres in 0.1, 0.3, 0.6, 0.9, and 1.2% concentrations with a diameter of 15 µm were purchased from Particle-works (Royston, United Kingdom). To generate the mechanical ultrasonic wave on the surface of a cell or a sphere, the acoustic tweezer system is required as shown in Fig. 3. The movement of the transducer was controlled by a three-axis motorized stage (SGSP 20, Sigma KOKI Co., Japan) and aligned the focal point on the mylar film using a pulser-receiver (5910PR; Olympus, Center Valley PA, USA). After alignment of a focal spot, 45 MHz sinusoidal burst signal, generated by function generator (Stanford Research Systems, Sunnyvale, CA, USA) and amplified by a 50-dB power amplifier (525LA, ENI, Rochester, USA) was driven on the transducer to grab and deform the suspended target. The duty cycle and PRF were set up as 450 cycles and 2 ms, respectively. The input peak-to-peak voltage was set as 12.6, 25.3, 38.0, 50.1, and 63.2 V. The acoustic trapping and cell deformation were observed by an inverted microscope (Olympus IX-71, Center Valley, PA, USA) and recorded via a CMOS camera (ORCA-Flash2.8, Hamamatsu, Japan). 5.2.5 Mechanical Test of Hydrogel Spheres Agarose hydrogel spheres in 0.1, 0.3, 0.6, 0.9, and 1.2% concentrations were mechanically tested using a micropipette aspiration technique (MAT). Micropipette aspiration is a well-known technique in mechanical measurement of a small sample such as a cell or a cell-sized particle. However, this technique requires the micropipette to be direct contact with the sample, which might cause a physical damage on the sample. For 80 this reason, a direct measurement on cells using a MAT was not preferable. A glass capillary with filament (GD-1, Narishige, NY) was heated in the middle by a vertical micropipette puller (PC-10, Narishige, NY). When the glass started to melt, the two halves of the glass are pulled apart to become micropipettes with the desired inner diameter. This micropipette was connected to a pressure controller (ez-gSEAL 100B, Neo biosystem, CA) in order to accurately generate the suction pressure in the range from 0 to 33.3 kPa. The suction pressure was controlled by an ez-Gseal control software (NBSC Controller, Neo biosystem, CA). As suction pressure from the micropipette turned on and increased, a hydrogel sphere attached to the tip of the micropipette and started to deform. The deformability was varied depending on its mechanical properties. Sphere deformation was observed using an inverted microscope and recorded via a CMOS camera. 81 Figure 5.3 Schematic diagram of experimental system. The SBAT at 45 MHz was driven by sinusoidal bursts from a function generator, amplified in a 50-dB amplifier. A single cell or a single sphere was deformed due to the SBAT. 5.2.6 Cell Viability Test Calcein-AM (Invitrogen, Carlsbad, CA, USA), a membrane permeable live-cell labeling dye, was used to examine the cell viability after the SBAT. The cell viability was analyzed based on the fluorescence level. The cells were cultured in PBS containing 1 µM Calcein-AM at room temperature for 30 minutes. The fluorescence level imaging of a Func%on'generator'&' 500dB'power'amp''' Microscope' &'CCD' camera' Cancer'cell'or' Hydrogel'sphere' Ultrasonic'transducer' Water' Mylar'film' Imaging' SoDware' XYZ'Motorized' Posi%oner' 82 targeted single cell (Ex: 480 nm, Em: 530 nm) was measured before and after the SBAT at 1.0 MPa. The mean and standard deviation of the fluorescence level with the sample size of 10 were calculated, and a two-tailed t-test with the level of significance of 1% was carried out. 5.3 Experimental Result 5.3.1 Deformation of Cell and Sphere under SBAT The SBAT has a capability to deform a trapped sample resulting in area changes along a transverse direction. During the SBAT on the cell or the sphere, their deformation was monitored and analyzed their area changes at indicated acoustic pressures. The pressure generated from the SBAT was gradually increased from 0 to 1.0 MPa. Driving condition of the SBAT was as follows: the number of cycles of 450, the PRF of 500 Hz and the input voltages to the transducer 0, 12.6, 25.3, 38.0, 50.1, and 63.2 Vpp (corresponding acoustic pressures: 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa). For cell deformation as a function of the acoustic pressure, three breast cancer cells: MDA-MD-231 (highly-invasive), MCF-7 (weakly-invasive), and SKBR-3 (weakly- invasive) were compared. As shown in Fig. 4 (a), it presents the comparison between the shape of three cells before and after trapping. Fig. 4 (b) represents the analysis of area changes of each cell line as a function of the acoustic pressure (sample number for each cell line:10). The area of the cell was computed using ImageJ. With boundary selection, the polygon was formed and its surface area was measured. It was found that the area of a cell was increased as the acoustic pressure was increased. It is clearly shown that all three 83 cell lines at 1.0 MPa were displayed a larger surface area compared to cells at 0 MPa in Fig. 4 (a) which means they are more deformed at higher acoustic pressures. The normalized area of the MCF-7 cells was measured to be 1.000, 1.027, 1.044, 1.062, 1.082, and 1.094 at 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa, respectively. The normalized area of SKBR-3 was found to be 1.000 at 0.00 MPa, 1.007 at 0.24 MPa, 1.027 at 0.43 MPa, 1.047 at 0.62 MPa, 1.075 at 0.81 MPa, and 1.089 at 1.00 MPa. A very similar tendency was observed for MCF-7 and SKBR-3 cells. In contrast, the normalized area of MDA-MD-231 cells was found to be considerably larger with measurements of 1.000, 1.030, 1.064, 1.084, 1.110, and 1.145 at 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa, respectively. The slopes of linear regression were calculated for each cell line by plotting normalized cell deformation-acoustic pressure, and found that the cells with higher slopes exhibited more deformable properties under the SBAT shown in Fig. 4. 84 (a) MCF$7& MDA$MB$231& SKBR$3& 0.95& 1& 1.05& 1.1& 1.15& 1.2& 1.25& 0& 0.2& 0.4& 0.6& 0.8& 1& 1.2& Normalized+deformability+ Acous4c+Pressure+(MPa)+ MDA$MB$231& MCF$7& SKBR$3& Before&SBAT& A<er&SBAT& 85 (b) Figure 5.4 Deformability of MDA-MB-231, MCF-7, and SKBR-3 cells. (a) Bright field images for each cell to show area changes before and after SBAT. Scale bars indicate 5 µm. (b) Normalized area changes of trapped cells at acoustic pressures of 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa. Error bars indicate standard deviations. Sample number for each cell line was 10. For sphere deformation under the SBAT, hydrogel spheres with different agarose concentrations (0.0, 0.3, 0.6, 0.9, and 1.2%) were employed. Since the mechanical properties of spheres are depended on their agarose concentration, they had different MCF$7& MDA$MB$231& SKBR$3& 0.95& 1& 1.05& 1.1& 1.15& 1.2& 1.25& 0& 0.2& 0.4& 0.6& 0.8& 1& 1.2& Normalized+deformability+ Acous4c+Pressure+(MPa)+ MDA$MB$231& MCF$7& SKBR$3& Before&SBAT& A<er&SBAT& 86 deformability under the SBAT. Fig. 5 shows the comparison on shape changes of the spheres for five different concentrations caused by the SBAT and their deformability as a function of the acoustic pressure (sample number for each concentration: 10). The normalized area of the 0.1% was measured to be 1.000, 1.223, and 1.312 at the acoustic pressure of 0.00, 0.24, and 0.43 MPa, respectively. Interestingly, the 0.1% sphere exploded at higher than 0.43 MPa. The normalized area of the 0.3% sphere was found to be 1.000, 1.070, 1.111, 1.134, 1.170, and 1.193 at 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa, respectively. The normalized area of the 0.6% was measured to be 1.000, 1.047, 1.065, 1.076, 1.083, and 1.095 at 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa, respectively. The normalized area of the 0.9% and 1.2% spheres at 1.00 MPa were 1.042 and 1.023, respectively. The slopes of linear regression were computed for each concentration by plotting normalized sphere deformation-acoustic pressure. As shown in Fig. 5 (b), the slope was inversely proportional to the concentration of agarose. The sphere with higher agarose concentration proved less likely to be deformed. Moreover, these results demonstrated that the SBAT induced more morphological deformation of the trapped cell or sphere with stronger acoustic pressure. 87 (a) 0.1%% 0.3%% 0.6%% 0.9%% 1.2%% 0.9% 1% 1.1% 1.2% 1.3% 1.4% 0.0% 0.2% 0.4% 0.6% 0.8% 1.0% 1.2% Normalized+deformability+ Acous4c+Pressure+(MPa)+ 0.10%% 0.30%% 0.60%%% 0.90%%% 1.20%%% (a)% (b)% Before%SBAT% A8er%SBAT% Before%SBAT% A8er%SBAT% 0.1%% 0.3%% 0.6%% 0.9%% 1.2%% 0.9% 1% 1.1% 1.2% 1.3% 1.4% 0.0% 0.2% 0.4% 0.6% 0.8% 1.0% 1.2% Normalized+deformability+ Acous4c+Pressure+(MPa)+ 0.10%% 0.30%% 0.60%%% 0.90%%% 1.20%%% (a)% (b)% Before%SBAT% A8er%SBAT% Before%SBAT% A8er%SBAT% 88 (b) Figure 5.5 Deformability of 0.1, 0.3, 0.6, 0.9, 1.2% agarose hydrogel spheres. (a) Bright field images for each sphere to show area change before and after SBAT. Scale bars indicate 5 µm. (b) Normalized area changes of trapped spheres at acoustic pressures of 0.00, 0.24, 0.43, 0.62, 0.81, and 1.00 MPa. Sample number of each concentration was 10. 5.3.2 Mechanical Properties Measurement using MAT In Fig. 6 it was observed that the deformation of a 0.3% agarose hydrogel sphere using a mechanical test, a MAT. As the suction pressure increased, the sphere was aspirated into the micropipette with increasing the corresponding aspirated length. For given values of the geometrical, rheological and external parameter, the Young’s modulus could be computed by the following expression (Theret et al., 1988): 𝑬= 𝟑𝑹∆𝚸 𝟐𝝅𝑫 𝝓(𝜼) (1) where E is the Young’s modulus, R is the inner radius of the micropipette, ΔP is the pressure difference, D is the corresponding aspirated length, and ϕ(η) is a wall function calculated using the punch model and was determined by the geometry of micropipette. Theret indicated that realistic value of η range from about 0.4 to 0.6 takes a mean ϕ(η) of ~2.05 (Sato et al., 1990; Theret et al., 1988). In this study, ϕ(η) was estimated to be mean value of 2.014 for the whole range of inner and outer micropipette fabricated. It should be noted that it is assumed a sphere is continuously deformable with isotropic and homogenous material properties. 89 (a) (1)$ (2)$ (3)$ (a)$ (b)$ 0.00$ 5.00$ 10.00$ 15.00$ 20.00$ 0.00$ 0.20$ 0.40$ 0.60$ 0.80$ 1.00$ 1.20$ Young's(Modulus((kPa)( Agarose(%( 90 (b) Figure 5.6 Mechanical test to investigate the Young’s modulus of 0.1, 0.3, 0.6, 0.9, 1.2% agarose hydrogel spheres. (a) A MAT was utilized to measure the mechanical properties of a 0.3% sphere. As suction pressure increased from the micropipette, the hydrogel sphere gradually deformed inside of the micropipette from (1) to (3). The red dotted line indicates how much the sphere was aspirated. Scale bars indicate 5 µm. (b) Measured young’s modulus for each sphere. Error bars indicate standard deviations. Sample number for each concentration was 10. The Young’s modulus of 0.1, 0.3, 0.6, 0.9, and 1.2% sphere was calculated to be 0.21 kPa (standard deviation of 0.08), 2.17 kPa (standard deviation of 1.08), 3.58 kPa (standard deviation of 1.49), 5.54 kPa (standard deviation of 2.28), and 11.35 kPa (standard deviation of 3.83), respectively, as shown in Fig. 5 (b). It reveals that the Young’s modulus was directly proportional to the amount of agarose in the sphere. Moreover, these results are in agreement with those previously measured in another (1)$ (2)$ (3)$ (a)$ (b)$ 0.00$ 5.00$ 10.00$ 15.00$ 20.00$ 0.00$ 0.20$ 0.40$ 0.60$ 0.80$ 1.00$ 1.20$ Young's(Modulus((kPa)( Agarose(%( 91 mechanical testing, Instron (Walker et al., 2011). With the findings of the Young’s modulus of each hydrogel sphere, I quantified the elastic properties of three breast cell lines. By interpolating the Young’s modulus of spheres into the slopes of cells in Fig. 4 and 5 (a MDA-MB-231 = 0.143, MCF-7 = 0.096, and SKBR-3 = 0.095) and spheres (0.1% = 0.735, 0.3% = 0.189, 0.6% = 0.088, 0.9% = 0.040, and 1.2% = 0.021), the Young modulus of three breast cancer cell lines could be indirectly estimated to be 1.59 ± 0.72 kPa for MDA-MB-231, 2.08 ± 0.85 kPa for MCF-7, and 2.10 ± 0.83 kPa for SKBR-3. 5.3.3 Cell Viability Test A cell viability test of breast cancer cells including MDA-MB-231, MCF-7, and SKBR-3 was performed using Calcein-AM. Fig. 8 (a) shows images of cell viability: positive control (treated with 0.1% bleach), control (before trapping), and negative control (after trapping). The normalized mean fluorescence that represents the live-cell dye was shown in Fig. 8 (b) with the sample number of 10 for each cell line. The normalized mean viability after trapping for MDA-MB-231, MCF-7, and SKBR-3 cells was found to be 0.97 with a standard deviation of 0.02, 0.95 with a standard deviation of 0.03, and 0.97 with a standard deviation of 0.02, respectively. There was no significant difference between the control group and the trapping group as the p-value of all three cell groups greater than 0.05 (MDA-MB-231 of 0.55, MCF-7 of 0.36, and SKBR-3 of 0.69). Consequently, trapping performance on cells did not induce any significant effect to the condition of cells under the indicated driving conditions. 92 (a) MDA$MB$231) MCF$7) SKBR$3) Posi4ve)Control) Nega4ve)Control) Control) 0)min) 0)min) 60)min) 120)min) 0)min) 0) 0.2) 0.4) 0.6) 0.8) 1) 1.2) control) 0)min) 60)min) 120)min) Normalized+Cell+Viability++ MDA$MB$231) MCF$7) SKBR$3) MDA$MB$231) MCF$7) SKBR$3) Nega4ve)Control) Control) 60)min) 120)min) 0)min) 0) 0.2) 0.4) 0.6) 0.8) 1) 1.2) control) 0)min) 60)min) 120)min) Normalized+Cell+Viability++ MDA$MB$231) MCF$7) SKBR$3) 93 (b) Figure 5.7 Cell viability test of MDA-MB-231, MCF-7, and SKBR-3 cells. (a) fluorescence images for a MDA-MB-231, a MCF-7, and a SKBR-3 cell for 0.1% bleach test (positive control), before SBAT (control), and after SBAT (negative control). Scale bars indicate 5 µm. (b) Normalized fluorescence intensity of cells before and after SBAT. Error bars indicate standard deviations. Sample number for each cell was 10. 5.4 Discussion This study demonstrated the quantification of mechanical properties of a single cell using the SBAT. It was discovered that the SBAT has a capability to deform the trapped particle or cell (Hwang et al., 2016). The feasibility of the SBAT to deform the cell and to investigate their characteristics of invasiveness was previously studied (Hwang et al., 2016; Hwang et al., 2014a). Although Hwang et al. offered the relative deformability and cell invasiveness, they were unable to measure the absolute value of mechanical properties such as the Young’s modulus (Hwang et al., 2016). This extended study could obtain the Young’s modulus of a single cell by developing cell-mimicking 0" 0.2" 0.4" 0.6" 0.8" 1" 1.2" control" 0"min" 60"min" 120"min" Normalized+Cell+Viability++ MDA4MB4231" MCF47" SKBR43" 94 phantoms with known mechanical properties. A cell-sized hydrogel sphere was constructed to function as a surrogate cell, exploring the feasibility of a deformed relation between a cell and a hydrogel sphere under the SBAT. The mechanical properties of spheres were measured by the MAT and allowed for indirect measurement of cell elasticity. One of the main advantages of the SBAT is to generate the acoustic pressure in broad ranges from kilo to megapascals. By controlling the driving condition of the SBAT, cell or sphere deformation could be clearly observed due to an acoustic trap at the given acoustic pressures. As a result, the absolute young’s modulus of cancer cells elucidated the metastasis potential of tumor cells. Biophysical methods to predict and estimate the cell stiffness are widely used under investigation to understand cellular process linked with diseases. Also, comparative analysis of cancer cell mechanics is a meaningful study to disease prognosis. In this study, I investigated three breast cancer cells with different degrees of malignancy: MDA-MB- 231, MCF-7, and SKBR-3 cells. Previously, direct measurements of breast cancer cells stiffness, including AFM and OT, have been developed. Calzado-Martin et al. reported that the Young’s modulus of MDA-MB-231 cells of 25 kPa in comparison to MCF-7 cells was 28 kPa (Calzado-Martín et al., 2016). Nikkhah et al. found the Young’s modulus of MDA-MB-231 and MCF-7 cells to be 0.4 kPa and 0.7 kPa, respectively (Nikkhah et al., 2011). Wang et al. estimated the Young’s modulus using AFM as MDA- MB-231 cells of 1.0 kPa, MCF-7 cells of 2.8 kPa, and SKBR-3 cells of 1.9 kPa (Wang et al., 2015). In general, the Young’s modulus of these cells obtained by the OT was a few orders of magnitude lower than the AFM. This was because parameter including probe 95 stiffness, loading rate and indentation force caused to affect on the measurement (Coceano et al., 2016). MDA-MB-231, MCF-7, and SKBR-3 cells typically exhibited different mechanical properties (Coceano et al., 2016; Nikkhah et al., 2011; Wang et al., 2015), and among them, MDA-MB-231 is the most aggressive cancer cell with high metastatic potential. On the other hand, MCF-7 and SKBR-3 cells are less-malignant cancer cell lines with low metastatic potential. I measured the mechanical properties of individual three cells using the SBAT as an alternative tool, the SBAT and proved that the average stiffness of MDA-MB-231 cells was much lower than the other two cells, which suggests that the results are in agreement with those previously measured by other methods (Calzado-Martín et al., 2016; Nikkhah et al., 2011; Wang et al., 2015). As mentioned above, the OT has been popularly used for mechanical characterization because it doesn't require any mechanical contact force with high force resolution. The OT and the optical stretcher have been used dual identical laser beams to trap two beads attached to opposite side of a cell and then to stretch it by manipulation. However, the optical laser power of a few Watts and the optical trapping force of up to a few piconewtons might not enough to directly deform a trapped cell. In contrast, since the SBAT can generates stronger trapping force up to a few hundred nanonewtons trapping force, it can push and squeeze the cell to deform along the transverse axis. By controlling for the driving condition of the SBAT, acoustic pressures could be adjustable depending on the stiffness of samples to trap and deform. In the present study, the acoustic pressure less than 1.0 MPa was enough for deforming breast cancer cells and cell-mimic spheres to distinguish. In the case of 0.1% hydrogel spheres, they started to burst out when the 96 acoustic pressure reached to 0.43 MPa, so it was not able to measure further. In the other hand, as proved in the cell viability test, the acoustic pressure of 1.0 MPa did not induce any effect to the cell condition. 5.5 Concluding Remarks I ascertained that the SBAT was another great tool for quantification of mechanical properties of a single cell with non-contact and non-label. With trapping force generated from the ultrasonic transducer, it was capable to capture and deform a cell or a sphere. Agarose hydrogel sphere functioned a surrogate cell and provided standardized reference data of cell elasticity. The elastic properties of hydrogel spheres were mechanically measured using the MAT. Based on the measurements of deformability of cells and spheres in addition to the Young’s modulus of sphere, the mechanical properties of the cell were acquired. In the last decades, investigation on mechanical properties of a cell has been issued for a better understanding of cell response and function in the circulation. Moreover, measurements of cancer cell elasticity to estimate its metastatic potential may be critical for treatment the disease. Previous studies measured the relative Young’s modulus of breast cancer cell using the SBAT and determined the invasion potential of cells (Hwang et al., 2016). In this study, I extended their work to measure absolute mechanical properties of a cell by using the SBAT. I calibrated the deformability of a cell by developing cell-mimicking spheres with the known Young’s modulus. This enabled us to indirectly quantify the absolute mechanical properties of cells. The drug treatment is highly correlated with mechanical properties of a cancer cell for controlling its metastatic potential. In future work, comparative analysis 97 of mechanical properties using the SBAT between treated and untreated cell lines will be dedicated for providing a better understanding of drug efficiency on their invasion potential. . 98 CHAPTER 6 Summary and Future Works 6.1 Summary This dissertation presented the quantification of cellular properties by the SBAT, a tool for non-contact assessment. To overcome the current frequency limit of SBATs, UHF transducers were fabricated for trapping a single cell and a cell-sized microparticle. In addition, to allow the precise control of a cell and investigate the cell mechanics, a quantitative knowledge of an acoustic trapping force is necessary. However, previous calibration techniques were not applicable for an UHF SBAT because they have been found to suffer form large experimental error when the particle size becomes in a few micrometer levels. Therefore, I proposed a new calibration method, a MAT because its advantages of precise particle control and the huge measurement ranges of the forces. After I calibrated its trapping force of the SBAT, inter-erythrocyte forces were quantified based on the pre-calibrated trapping force. At first, I reported a method to calibrate the acoustic trapping force from the SBAT at UHF using a MAT. The acoustic trapping forces and the trap stiffness on a 5- µm polystyrene microbead for a 110-MHz SBAT were measured against the known force generated from a micropipette. The trap stiffness, which represents the trapping force corresponding to a displacement of a microbead from the trap center, was measured and the results showed that a higher duty factor and excitation voltage lead to a stronger trapping force and trap stiffness for a given displacement. Since a precisely calibrated force generated from a micropipette is directly applied to the calculation of acoustic 99 trapping force, the approach should be more accurate than those previously reported. In addition, with this method precisely controlling the tip size of a micropipette within a few micrometers allows more accurate calibration of the trapping force on an object of the size of a single cell. Efforts on quantitative measurements of the interactive forces of RBCs have been pursued for a better understanding of hemodynamics and blood rheology. I reported an approach based on an UHF SBAT for quantitative measurements of inter-RBC forces at a single cell level. Once again, the trapping forces produced by this UHF SBAT can be quantitatively estimated with a micropipette. Since the focal beam diameter of the 410 MHz ultrasonic transducer used in this SBAT was only 6.5 micrometer, which was smaller than that of a RBC, it was made possible to directly apply the beam to a single RBC and measure inter-RBC forces against the pre-calibrated acoustic trapping forces as another example of potential cellular applications of the SBAT. The magnitude of these forces was found to be 391.0 ± 86.4 pN. Lastly, the mechanical properties of cancer cells were quantified by the SBAT at 45 MHz. This transducer trapped a single suspended cell and elicited the morphological cell membrane deformation. Cell-mimicking phantoms, agarose hydrogel spheres in 0.1~1.2% concentrations served as the standardization of the biomechanical characteristics of cells. The single hydrogel sphere was trapped and deformed by the SBAT as well. Young’s modulus of a hydrogel sphere in each concentration was determined by a mechanical test using a MAT. Based on an analytical comparison of 100 deformability levels between the cell and the sphere, the indirect measurement of the mechanical properties of cells could be achieved by interpolating Young’s modulus of spheres. As a result, Young’s modulus of MDA-MB-231, MCF-7, and SKBR-3 cells was found to be 1.59 ± 0.72 kPa, 2.08 ± 0.85 kPa, and 2.10 ± 0.83 kPa, respectively. Results from these examinations indicated that highly invasive cancer cells exhibited softer mechanical properties than weakly-invasive cancer cells. This study demonstrated the feasibility of the SBAT for investigating biomechanical characteristics of a single cell without labeling. Even though investigating cellular properties is critical for disease diagnosis in a clinical field, not many experimental studies have been demonstrated, and their results are not consistent. As proved by experimental results in this dissertation, UHF SBATs serve as a novel tool for trapping a single cell and determining cellular properties. In near future, I are promised that the valuable quantification studies offered from this paper will play a vital role on biomechanical information for clinical diagnoses. 6.2 Future Works Recently, acoustic levitation for microparticle manipulation have gained considerable attention for biomedical applications. For this reason, many manipulation methods including standing wave and surface acoustic wave have developed the three- dimensional trapping. Acoustic standing waves consist of two opposite acoustic waves which generates the nodes, where a particle is trapped (Ding et al., 2012). More recently, an acoustic trap was shown to be capable of levitating one particle in three-dimensions 101 using the negative gradient forces in the low megahertz range as shown in Figure 6.1. (Baresch et al., 2016). Figure 6.1 Sketch of the particle trapping in three dimensions. The beam is focused upon polystyrene particles lying on an acoustically transparent (Baresch et al., 2016). The SBAT is an excellent tool for selectively manipulating objects with a huge range of the particle size, but only two-dimensional trapping by the SBAT is possible at present. This is because single-side trapping devices need more complicated setup for acoustic levitation than that of opposite-side devices. Present two-dimensional SBAT is only possible when a targeted particle is placed above a membrane such as a mylar film, so that the trapped particle stayed on a plane of the membrane due to the scattering force. For three-dimensional SBAT, it is required to generate a negative pulling force opposite 102 to the propagation direction to move the particle back to the transducer. 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Abstract (if available)
Abstract
Recently, a single-beam acoustic tweezer (SBAT) has successfully been developed for numerous biomedical applications involving trapping macromolecules and cells. Yet even with its extensive improvement, it was still difficult to apply previous ultrasonic tweezer techniques to study mechanical properties of a single cell due to the frequency limit. ❧ To overcome the previous frequency limit, fabrication of ultra-high frequency (UHF) single-element ultrasonic transducers (>100-MHz) was proposed. As these transducers’ beam width is inversely proportional to the center frequency of the transducer, the proposed transducers are able to manipulate a biological cell and a cell-sized microparticle. Also, quantitative knowledge of the trapping forces from the SBAT is necessary to allow the precise control of cell manipulation and to quantify cell mechanics. Therefore, I first calibrated the trapping forces of the UHF SBAT using a micropipette aspiration technique (MAT). As a result, calibrated trapping forces were found to exhibit a wide range, from the piconewton to nanonewton level, which is the strongest among the micromanipulation technologies. ❧ Quantification of cellular properties has been studied for cell regulation and function. I developed a method to quantify inter red blood cell (RBC) forces using SBATs with a pre-calibrated trapping force. The previous SBATs have been used to attempt to demonstrate the quantification of intercellular events but did not get meaningful results because there were limitations to frequencies. However, a newly fabricated 410 MHz UHF transducer allowed the quantitative study of cell-cell interactions by producing a beam size of a few micrometers. In addition, the quantification of mechanical properties of suspended human breast cancer cells was demonstrated by a non-mechanical contact SBAT at high frequency. I obtained absolute properties of the cell membrane to determine the metastatic potential of cancer cells in vitro. Results from these examinations could demonstrate the feasibility of the SBAT, a non-destructive assessment tool of clinical diagnosis for investigating biomechanical characteristics of cells.
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Creator
Lim, Hae Gyun
(author)
Core Title
Quantification of cellular properties using ultra-high frequency single-beam acoustic tweezer
School
Viterbi School of Engineering
Degree
Doctor of Philosophy
Degree Program
Biomedical Engineering
Publication Date
04/25/2018
Defense Date
10/17/2017
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University of Southern California
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Tag
breast cancer cell,calibration of trapping force,cell mechanics,OAI-PMH Harvest,red blood cell,single-beam acoustic tweezer,ultra-high frequency ultrasonic transducer
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English
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Electronically uploaded by the author
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Shung, Koping Kirk (
committee chair
), Shen, Keyue (
committee member
), Zhou, Qifa (
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haelim@usc.edu,jjangas3@hotmail.com
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Lim, Hae Gyun
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Tags
breast cancer cell
calibration of trapping force
cell mechanics
red blood cell
single-beam acoustic tweezer
ultra-high frequency ultrasonic transducer