The Pennsylvania State University The Graduate School College of Engineering SIMULATIONS FOR INVESTIGATING THE CONTRAST MECHANISM OF BIOLOGICAL CELLS WITH HIGH FREQUENCY SCANNING ACOUSTIC MICROSCOPY A Dissertation in Acoustics by Yada Juntarapaso 2017 Yada Juntarapaso Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2017 The dissertation of Yada Juntarapaso was reviewed and approved* by the following: Richard L. Tutwiler Associate Professor of Acoustics Dissertation Adviser Chair of Committee Victor W. Sparrow Professor of Acoustics Interim Chair of the Graduate Program in Acoustics Thomas B. Gabrielson Professor of Acoustics Margaret J. Slattery Assistant Professor of Bioengineering *Signatures are on file in the Graduate School iii ABSTRACT Scanning Acoustic Microscopy (SAM) is one of the most powerful techniques for nondestructive evaluation and it is a promising tool for characterizing the elastic properties of biological tissues/cells. Exploring a single cell is important since there is a connection between single cell biomechanics and human cancer. Scanning acoustic microscopy (SAM) has been accepted and extensively utilized for acoustical cellular and tissue imaging including measurements of the mechanical and elastic properties of biological specimens. SAM provides superb advantages in that it is non-invasive, can measure mechanical properties of biological cells or tissues, and fixation/chemical staining is not necessary. The first objective of this research is to develop a program for simulating the images and contrast mechanism obtained by high-frequency SAM. Computer simulation algorithms based on Matlab® were built for simulating the images and contrast mechanisms. The mechanical properties of HeLa and MCF-7 cells were computed from the measurement data of the output signal amplitude as a function of distance from the focal planes of the acoustics lens which is known as V(z) . Algorithms for simulating V(z) responses involved the calculation of the reflectance function and were created based on ray theory and wave theory. The second objective is to design transducer arrays for SAM. Theoretical simulations based on Field II© programs of the high frequency ultrasound array designs were performed to enhance image resolution and volumetric imaging capabilities. Phased array beam forming and dynamic apodization and focusing were employed in the simulations. The new transducer array design will be state-of-the-art in improving the performance of SAM by electronic scanning and potentially providing a 4-D image of the specimen. iv TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………………..........vii LIST OF TABLES…………………………………………………………………………....xvii ACKNOWLEDGEMENTS…………………………………………………………………..xviii Chapter 1 Introduction................................................................................................................1 1.1 Background and motivation........................................................................................ 1 1.2 Specific aims .............................................................................................................. 2 1.3 Dissertation outline .................................................................................................... 3 Chapter 2 Background in Understanding Scanning Acoustic Microscopy .............................. 5 2.1 Principles of Scanning Acoustic Microscopy ............................................................ 5 2.1.1 Acoustic lens ................................................................................................... 7 2.1.2 Coupling medium ............................................................................................ 9 2.1.3 Resolution of the acoustic microscope ............................................................ 10 2.1.4 Imaging procedure ........................................................................................... 12 2.2 Examples of commercially available SAMs .............................................................. 13 2.3 Applications of SAM in pulse mode (time-resolved scanning acoustic microscopy) .................................................................................................................................. 17 2.4 Applications of SAM in tone burst mode (V(z) technique) ....................................... 18 2.5 Explore biological cells and tissue with SAM ........................................................... 19 2.5.1 Investigating cells in culture with SAM..........................................................19 2.5.2 Investigating biological specimens with time-resolved SAM ......................... 22 Chapter Summary..............................................................................................................22 Chapter 3 Background in Understanding V(z) curve...............................................................23 3.1 The V(z) curve ........................................................................................................... 23 3.2 Theory of V(z) ........................................................................................................... 25 3.2.1 Ray models ...................................................................................................... 25 3.2.2 Fourier Angular Spectrum Theory or Wave Theory model ............................ 29 Chapter Summary.............................................................................................................42 Chapter 4 Background in Understanding Biological Cell........................................................ 43 4.1 Contrast mechanism for acoustic imaging of biological cells and tissues ................. 44 4.2 Layer model and reflectance function of biological cells .......................................... 46 4.3 Mechanical properties measurements for single cells and biological tissues............. 54 4.3.1 Mechanical properties measurement by time-resolved SAM ......................... 55 v 4.3.2 Time-resolved techniques and elastic microanalysis ..................................... 58 4.4 HeLa cells .................................................................................................................. 67 Chapter Summary...............................................................................................................74 Chapter 5 Background in Understanding Ultrasound Transducer Array ................................. 75 5.1 Transducers and Arrays.............................................................................................. 78 5.1.1. Single element transducer .............................................................................. 80 5.1.2 Arrays .............................................................................................................. 83 5.2 Focusing ..................................................................................................................... 89 5.2.1 Axial and Lateral Resolution ........................................................................... 89 5.2.2 Focusing .......................................................................................................... 96 5.3 Ultrasound Imaging .................................................................................................... 98 5.3.1 B-scan .............................................................................................................. 99 5.3.2 C-Scan ............................................................................................................. 101 Chapter Summary..............................................................................................................101 Chapter 6 Methods and Simulations ........................................................................................ 102 6.1 Calculation of reflectance function for biological cell and substrate ......................... 102 6.2 Simulations of V(z) curves......................................................................................... 103 6.2.1 FFT analysis of V(z) curves ............................................................................ 106 6.3 Extracting the mechanical properties of biological cells from time-resolved SAM .. 113 6.3.1 Acoustical images and data acquisition ........................................................... 113 6.3.2 Thickness, sound velocity, and attenuation of cells ........................................ 117 6.4 Transducer Simulations .............................................................................................. 120 6.4.1 Point targets ..................................................................................................... 121 6.4.2 Cyst phantom................................................................................................... 122 6.4.3 Cell model ....................................................................................................... 122 Chapter Summary..............................................................................................................123 Chapter 7 Simulation Results ................................................................................................... 124 7.1 Reflectance function for biological cells and substrate .............................................. 125 7.1.1 Fused quartz substrate ..................................................................................... 125 7.1.2 Kidney tissue 3 µm thickness on fused