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Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zeeb Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 EFFECT OF MYOFIBRIL LENGTH AND TISSUE CONSTITUENTS ON ACOUSTIC PROPAGATION PROPERTIES OF MUSCLE BY NADINE BARREE SMITH B.S., University of Illinois, 1985 M.S., University of Illinois, 1989 THESIS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics in the Graduate College of the University of Illinois at Urbana-Champaign, 1996 Urbana, Illinois UMI Number: 9702669 UMI Microform 9702669 Copyright 1996, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 © Copyright by Nadine Barrie Smith, 1996 UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN THE GRADUATE COLLEGE MAY 1996 WE HEREBY RECOMMEND THAT THE THESIS BY NADINE BARRIE SMITH FYTTTT.F.n EFFECT OF MYOFIBRIL LENGTH AND TISSUE CONSTITUENTS ON ACOUSTIC PROPAGATION PROPERTIES OF MUSCLE BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF_ DOCTOR OF PHILOSOPHY lt>$7L~s Head of Department Committee on Final Examination! PU-'L & i Chairperson /,)-0j§i^ t Required for doctor's degree but not for master's. O-SIT Ill EFFECT OF MYOFIBRIL LENGTH AND TISSUE CONSTITUENTS ON ACOUSTIC PROPAGATION PROPERTIES OF MUSCLE Nadine Barrie Smith, Ph.D. Graduate Program in Biophysics University of Illinois at Urbana-Champaign, 1996 Professor William D. O'Brien, Jr., Advisor The ultrasonic speed and attenuation coefficient of three muscle samples were measured in-vitro as a function of temperature and frequency. The sarcomere length of each of these samples was also determined. The three muscle groups were bovine longissimus dorsi, psoas major and lobster extensor. These were chosen to separately determine the effects of physical components (% water, % fat and % protein) and structural components (sarcomere length) on the ultrasonic propagation properties. The scanning laser acoustic microscope (SLAM) was used to measure the attenuation coefficient, speed and heterogeneity index of all three samples at 100 MHz. The Daedal exposimetry system (DES) was used to determine both of these quantities as a function of frequency over a range 2-7 MHz. In addition, measurements were made at three temperatures, 4, 20 and 37°C. The chemical composition of the muscle groups was determined using wet/dry techniques (% water), ether extraction (% fat) and nitric acid digestion (% protein). The sarcomere length for individual samples was determined using light microscopy of suitable stained slides. Finally, the effect of the sarcomere structure was determined by homogenizing the tissue samples, and measuring ultrasonic propagation. The destruction of the regular myofibril architecture showed that the muscle structure plays a critical role in the propagation of ultrasound through tissue. Using the results from these studies a series of multiple linear regression models was determined, which allowed the physical and structural determination of a fourth muscle type (bovine semitendinosus) based solely on the acoustic properties. Subsequent measurement of the physical and structural properties were made to test the accuracy of the developed models. The prediction model for estimating tissue constituents and sarcomere lengths at 100 MHz is remarkably accurate, with very low errors in estimating the tissue constituents within 5% and sarcomere lengths with an accuracy of less than half a micron. Overall, the empirical model developed using the DES was not as accurate as that of the SLAM. Results at the lower temperature of 4°C were consistently better than those at higher temperatures, with all parameters being predicted within 10% of the measured values. iv DEDICATION To my mother and father. V ACKNOWLEDGEMENTS I would like to most gratefully thank Professor William D. O'Brien, Jr. for giving me constant encouragement, endless understanding and support over the years. His friendship, belief and patience has enabled me to get this far. I will always be grateful to him for giving me the many opportunities to travel and to present this and other research at many conferences. This project could not have been completed without the use of the biochemical analysis facilities of Dr. Neal Merchen and the patience and lessons of Laura Bauer. Additionally, I could not have made the sarcomere length measurements without the help and time of my good friend, Dr. Jim Zachary. My gratitude goes out to Dr. Philip Best and Dr. Albert Feng for their helpful comments on my thesis and their willingness to serve on my committee. Throughout my career at the Bioacoustics Research Laboratory I have benefited from the friendships and camaraderie of many people built on the days of working and struggling together. On many an occasion I have run to others who have helped me out of difficult situations. Those times I will never forget (i.e. laboratory moving, chasing loose chinchillas, bulls chasing me, flooding basements, searching in skids for lost data, etc. etc.). I would like to acknowledge Wanda Elliot for always having an ear that I could bend; to Billy McNeil who would fix everything in a jiffy and always have the best sense of humor; to Bob Cicone who would fix my sick computer and be a friend that I could talk to about fishing. I will always cherish the friendship of Lisa Wilmes who helped me survive the early years of the Ph.D. program. Within my group I would like to recognize my "brothers and sisters" who have been there through thick and thin; Eric Chen, Scott Ellis, Rich Czerwinski, Kate Hillsley and Dudley Swiney. Special thanks to Amy Anderson for her constant assistance and making this process so much easier by her presence. And finally, I gratefully acknowledge the unfailing aid and constant emotional reassurance of Andrew Webb. vi TABLE OF CONTENTS CHAPTER PAGE 1. INTRODUCTION 1 2. ACOUSTIC THEORY 5 3. SKELETAL MUSCLE PHYSIOLOGY 26 4. ULTRASONIC PROPAGATION PROPERTIES OF BIOLOGICAL MOLECULES AND MUSCLE 37 5. INSTRUMENTATION 43 6. METHODS 48 7. RESULTS 65 8. DISCUSSION 83 APPEND LX A FIGURES 90 APPENDLX B TABLES 139 BIBLIOGRAPHY 174 VITA 182 vii LIST OF TABLES TABLE PAGE 3.3.3-1 Collagen types in tissue 140 3.3.4-1 Myofibril proteins of skeletal muscle 141 3.3.5-1 Thirteen bovine skeletal muscles listed by anatomical name 142 3.3.5-2. Rank of thirteen muscles by tissue constituents 143 4.1-1 Propagation properties for tissues of various collagen content. 144 4.2-1 Literature survey of bovine skeletal muscle 145 7.1.2-1. Speed of sound Dow Corning 710 for the SLAM 148 7.2.1-1 Attenuation coefficient of Dow Corning 710 for the DES 148 7.2.2-1 Speed of sound Dow Corning 710 for the DES 149 7.2.2-2 Summary of the uncertainty assessment for the SLAM and DES 149 7.3.2-1 The factor levels for each factor included in the ANOVA 150 7.3.2.1-1 ANOVA for orientation on the attenuation coefficient 150 7.3.2.1-2 Attenuation coefficient at 100 MHz for the three muscle types 150 7.3.2.2-1 ANOVA results for orientation and thickness factors on the speed 151 7.3.2.2-2 Speed results for the three muscle types 151 7.3.2.3-1 ANOVA results for orientation and thickness factors on the HI 152 7.3.2.3-2 Heterogeneity index results for the three muscle types 152 7.3.3-1 Biochemical analysis and sarcomere lengths for the three muscle types 153 7.3.3-2 Pearson coefficient and significance results 153 7.3.3-3 T-test results of the sarcomere lengths and tissue constituents 154 7.3.4-1 Regression equation between the acoustic properties and the physics characteristics 155 7.3.4-2 Linear regression results on the pooled acoustic data. 156 7.3.5-1 Multiple linear regression equations for the tissue constituents and the sarcomere length 157 viii TABLE PAGE 7.3.5.1-1 ANOVA was performed to assess the effect of the orientation factor on the attenuation coefficient response variable 158 7.3.5.1-2 Attenuation coefficient for bovine semitendinosis 158 7.3.5.1-3 ANOVA results of the factors on the speed for semitendinosis 158 7.3.5.1-4 Speed results for the bovine semitendinosis 158 7.3.5.1-5 ANOVA results for orientation and thickness for the HI 159 7.3.5.1-6 Heterogeneity index results for semitendinosus 159 7.3.5.2-1 Biochemical analysis and sarcomere lengths results of semitendinosis.