Approved as to style and content by:

____________________________ ____________________________

Gerard L. Coté Steven M. Wright

(Co-Chair of Committee) (Co-Chair of Committe)

____________________________ ____________________________

Sohi Rasteger Jeremy S. Wasser

(Member) (Member)

____________________________ ____________________________

Hsin-i Wu Way Kuo

(Member) (Head of Department)

An optical fluorescence spectrometer for measurement of intracellular calcium concentration and a double-tuned ³¹P/ ¹H radio frequency coil were constructed to allow for simultaneous measurement of optical fluorescence and ³¹ P nuclear magnetic resonance (NMR) spectra. The combination of these modalities not only increases the amount of information which can be learned from a single experiment since NMR sensitive and fluorescence sensitive variables may be monitored together, but also reduces subject and treatment variability since only a single experiment is performed. The utility of this technique was demonstrated by simultaneously measuring intracellular calcium concentration (via optical fluorescence) and high-energy phosphate metabolic state (via NMR spectroscopy) in the in vivo, in situ heart of the western painted turtle Chysemys picta bellii . This species is interesting to comparative cardiac physiologists because of its remarkable tolerance to prolonged and severe hypoxia. Additionally, techniques including the use of appropriately designed coil arrays were investigated as a means to maximize the signal-to-noise ratio over a particular volume of interest in an NMR experiment.

This work would not have been remotely possible without the support, direction, and understanding of many persons, and probably God too. My patient parents Pat and Roger and not so patient wife Alicia top the list. My committee members Gerry Coté, Steve Wright, Jeremy Wasser, Sohi Rastegar, and Wally Wu are next for providing invaluable guidance and support, unbelievable faith in me, and their signatures. I could not have done this without friends and role models like Marcel Goetz and Doug Macha, and the friendship and intellectual stimulation of co-workers like Jim Bankson, Dan Spence, Chris Gefrides, Mike McShane, Brent Cameron, Ashok Gowda, Brent Bell, and everyone else with whom I had the pleasure of working in the Optical Biosensing Lab, Magnetic Resonance Systems Lab, Biomedical Optics Lab, and Biomedical Laser Lab. I am indebted to Feng Xu who helped with many animal preparations, and I would like to thank my many other friends who helped me to accomplish the most difficult task of all: keeping my sanity during six years in College Station.

As a matter of personal principle, I have made this document to the greatest extent possible ``Microsoft-Free.'' This document was prepared and crafted almost entirely within the Linux operating system which is a free and publicly available flavor of Unix which runs on Intel-based PC's. The Applix ware office suite was used to prepare this thesis, and a substantial amount of the data analysis was performed using John Eaton's Octave which is also freely available. All of this was accomplished under the XFree86 windowing system. I also thank, then, the entire Linux development community for bringing to bear a powerful, usable, and responsive alternative MSW.

Page

ABSTRACT iii

DEDICATION iv

ACKNOWLEDGEMENTS v

TABLE OF CONTENTS vi

LIST OF FIGURES ix

LIST OF TABLES xii

CHAPTER

Chapter 1: INTRODUCTION AND OVERVIEW 1

1.1 Background 1

1.2 Motivation 4

1.3 Problem Statement 5

Chapter 2: CARDIAC PHYSIOLOGY 9

2.1 Introduction 9

2.2 Overview of the Cardiac System 9

2.3 Cardiac Mechanics 12

2.4 Excitation Contraction Coupling 13

2.5 Cardiac Metabolism 20

2.6 Measurements of Cardiac Function 24

Chapter 3: NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY 30

3.1 History of Magnetic Resonance 30

3.2 The NMR Signal 31

3.3 The NMR Coil 33

3.4 Design of a Coil Array for NMR Spectroscopy 39

3.5 Design of a Doubly-Tuned, Inductively Coupled Resonator 52

Page

CHAPTER

Chapter 4: OPTICAL FLUORESCENCE SPECTROSCOPY 58

4.1 Introduction to Fluorescence Spectroscopy 58

4.2 Fluorescent Metal Ion Indicators 59

4.3 Tissue Autofluorescence 62

4.4 Dye Choice 64

4.5 Instrument Design 66

4.6 Preliminary Experiments 70

Chapter 5: ANIMAL ANATOMY AND PREPARATION TECHNIQUES 73

5.1 Animal Selection 73

5.2 Gross Preparation 73

5.3 Pericardial Loading of fluo-3/AM 74

5.4 NMR Coil and Optical Fiber Placement 75

5.5 Unidirectional Ventilation Technique 76

Chapter 6: RESULTS 79

6.1 Introduction 79

6.2 Preliminary NMR Results 80

6.3 Preliminary Optical Fluorescence Results 81

6.4 Unidirectional Ventilation Experiments 85

6.5 Dual Spectroscopy Experiments 87

6.6 Experimental Protocol 88

6.7 Data Analysis 91

6.8 Dual Spectroscopy Results 103

6.9 Discussion of Dual Spectroscopy Results 107

Chapter 7: SUMMARY AND CONCLUSIONS 110

7.1 Summary 110

7.2 Conclusions 112

7.3 Future Work 114

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REFERENCES 118

Citations 118

Supplementary Sources 126

APPENDIX A 127

APPENDIX B 129

APPENDIX C 132

APPENDIX D 133

APPENDIX E 135

APPENDIX F 139

APPENDIX G 144

VITA 147

FIGURE Page

2.1 Typical Reptilian Heart from the Species Lacerta virdis 11

2.2 Schematic Representation of the Great Vessels

in the Reptilian Circulatory System 11

2.3 A Typical Cardiac Muscle Sarcomere 13

2.4 Excitation Contraction Coupling 15

2.5 Control of [Ca²⁺] Transients throughout the Cardiac Cycle 19

2.6 Energy Budget of the Heart 24

3.1 NMR Coil Match Network 38

3.2 Cross Section of Reptilian Heart and ROI 41

3.3 Use of an Array to Match FOV to ROI 42

3.4 Plot of SNR_rel versus Coil Diameter 48

3.5 Photograph and Drawing of Two-Chambered NMR Heart Phantom 49

3.6 Schematic of Inductively Fed Doubly-Tuned Resonator 54

3.7 Impedance Magnitude |Z_equiv| versus f for Doubly-Tuned Resonator 56

3.8 Plot of Probe Resonance from Network Analyzer 57

4.1 Jablonski Diagram Depicting the Optical Phenomenon

of Fluorescence 59

4.2 Schematic Representation of AM Loading 62

4.3 Chemical Structure of Calcium Chelator Dye fluo-3 65

4.4 Typical fluo-3 Fluorescence 65

4.5 Schematic of Optical Setup 68

FIGURE Page

4.6 Fiber Optic Coupler Detail 68

4.7 Photograph of Portable Fiber-based Optical Spectroscopy System 71

4.8 Photograph of Optical Fiber Chuck and Fiber Coupler 72

5.1 Relative Anatomy of the Turtle Chrysemys scripta 74

5.2 Dual Spectroscopy Preparation 76

5.3 Schematic of Unidirectional Breathing Apparatus 78

6.1 fluo-3 Calibration/Verification Experimental Setup 82

6.2 fluo-3 Spectra Obtained in Magnet Calibration Experiment 83

6.3 Real-time In vitro Fluorescence Data 85

6.4 Unprocessed Raw ³¹P FID 93

6.5 ³¹P FID after 15 Hz Line-broadening 93

6.6 Real, Imaginary, and Magnitude Components of Transformed FID 94

6.7 Phase-corrected Real ³¹P NMR Spectrum with Peak Assignments 94

6.8 In vivo fluo-3 Fluorescence with 10 sec Integration Time 99

6.9 In vivo fluo-3 Fluorescence with 0.1 sec Integration Time 99

6.10 Slope Corrected Fluorescence Intensity 100

6.11 Average of Slope Corrected Fluorescence Intensity 100

6.12 CCD Recorded In vivo Fluorescence Spectra 102

6.13 Calcium-dependent Fluorescence Intensity as % of Control 102

6.14 Mean-centered fluo-3 CCD Fluorescence Spectra 104

FIGURE Page

6.15 Stack Plot of In vivo ³¹P NMR Spectra 105

6.16 Relative Levels of PCr and b-ATP as % of Control 105

6.17 Simultaneously Recorded In vivo Fluorescence Spectra 106

6.18 Relative [Ca²⁺]-dependent Fluorescence Intensity as % of Control 106

6.19 Relative Levels of PCr, b-ATP, and [Ca²⁺ ] during Anoxia 109

D.1 NMR Spectroscopy Pulse Sequence 134

F.1 Successive ³¹P NMR Scans from In vivo Turtle Heart 140

F.2 Relative Levels of PCr and b-ATP from Figure-F.1 as % of Control 141

F.3 Successive ³¹P NMR Scans from In vivo Turtle Heart

with Unidirectional Breathing 142

F.4 Relative Levels of PCr and b-ATP from Figure-F.3 as % of Control 143

TABLE Page

3.1 Turtle Plasma Composition 46

3.2 Calculated SNR Values 47

3.3 Measured SNR Values 51

3.4 Resulting Doubly-Tuned Resonator Design 55

4.1 Some Common Fluorescent [Ca²⁺] Indicators 60

6.1 Assessment of Double-Tuned Resonator 80

6.2.a Blood Gas Measurements during Unidirectional Ventilation 86

6.2.b Blood Gas Measurements during Tidal Ventilation 87

C.1 TES Ringer's Solution Composition 132