CO-REGISTRATION OF BIOLUMINESCENCE TOMOGRAPHY AND ANATOMICAL IMAGING MODALITIES FOR CELL TRACKING AND SOURCE QUANTIFICATION by Moussa Chehade A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, Maryland May, 2014 © 2014 Moussa Chehade All Rights Reserved Abstract Bioluminescence tomography (BLT) is a molecular imaging tool that provides three-dimensional, quantitative reconstructions of bioluminescent sources in vivo. A main limitation of BLT to date, however, has been a lack of validation and demonstrated utility in preclinical research. An approach employing a fusion of BLT with other, well-established imaging modalities was used in this work to validate results obtained with BLT and improve the performance of source quantification. In the first chapter of this thesis, a method was developed to co-register BLT to magnetic resonance (MR) and computed tomography (CT) anatomical data for tracking cell transplants using a specialized animal holder. Using a luciferase- expressing tumor model in mice, MRI was shown to be superior at locating cells while BLT provided a more sensitive measure of cell proliferation. A multimodal approach incorporating BLT can therefore provide a better understanding of cell dynamics in vivo in preclinical research than with anatomical imaging alone. In the second chapter of this thesis, anatomical MRI and CT images were segmented to provide hard spatial priors to quantify the power of calibrated luminescent sources implanted in mice. To do this, a finite element (FEM) implementation of the diffusion approximation was used as a forward model for light propagation and validated through a phantom experiment. Source powers quantified using hard prior information showed a 65% reduction in average ii deviation compared to traditional BLT using four spectral bins and comparable performance to eight bins. BLI imaging times using hard spatial priors were reduced by 16-fold and 100-fold compared to the four- and eight-bin BLT methods, respectively. Together with the results of the first chapter, these results show value in incorporating data from other imaging modalities into BLT. Thesis Committee: Jeff W. M. Bulte, Professor of Biomedical Engineering, Thesis Advisor Piotr Walczak, Associate Professor of Radiology Kevin J. Yarema, Associate Professor of Biomedical Engineering iii Acknowledgements The numerous medical and pre-clinical imaging modalities available today are a testament to the creativity of the individuals working in this research field, one that I have been fortunate enough to be a part of over the past two years. I would like to take this opportunity to thank a very talented group of individuals for their support; this thesis would not have been possible without them. First and foremost, I would like to thank my advisor, Jeff W. M. Bulte, for his continuous guidance and support during my graduate studies. Dr. Bulte’s emphasis on treating all members of the research group, regardless of experience, as independent scientists was intimidating to me at first, but in retrospect a tremendous help in developing my skills as a researcher. I am also thankful for the support of Piotr Walczak, who provided valuable suggestions on my work and for the opportunities he provided me with to improve my skills in image analysis and visualization. I would also like to thank Kevin Yarema for his valuable feedback on this thesis. Very special thanks go to Amit Srivastava for his mentorship over the past two years. Amit has taught me much of what I know in wet lab skills and was always willing to sacrifice his time and effort to assist me with experimentation. I am indebted to him for his help. I would like to acknowledge the help of Irina Shats for sharing her wisdom in and training me in cryosectioning and staining protocols. A thanks goes out to Lisa Song for her guidance on the relevant literature and software for bioluminescence tomography. I would also like to acknowledge Antje Arnold and iv Anna Jablonska for their advice in day-to-day issues that came up during the course of my graduate work and their always appreciated sense of humor. To my lab mates and others who I may have forgotten, I am grateful to you for your willingness to help and your friendship. It has been a pleasure working with you all. Last but not least, I would like to extend a heartfelt thanks to my parents for their unconditional support and words of encouragement during my time at Johns Hopkins. Without them I would not have gotten this far, and for that I am truly grateful. v Table of Contents Abstract ...................................................................................................... ii Acknowledgements ..................................................................................... iv Table of Contents ...................................................................................... vi List of Tables ............................................................................................. ix List of Figures ............................................................................................. x Introduction ................................................................................................ 1 Chapter 1: Bioluminescence Tomography and MRI for Cell Tracking ........ 3 1.1 Background ................................................................................... 3 1.1.1 Bioluminescent Imaging ..................................................... 3 1.1.2 Limitations of Planar BLI .................................................. 4 1.1.3 Bioluminescence Tomography ............................................ 7 1.1.4 Alternative Cellular and Molecular Imaging Modalities ... 10 1.1.5 Co-registration and Image Fusion .................................... 12 1.1.6 Approach and Significance of this Work .......................... 13 1.2 Methodology and Animal Holder Design ..................................... 14 1.2.1 Equipment ........................................................................ 14 1.2.2 Imaging Workflow ............................................................ 14 1.2.3 Holder Requirements ........................................................ 15 1.2.4 Holder Design ................................................................... 16 1.3 Phantom Tests and Co-Registration Procedure .......................... 19 1.3.1 Phantom .......................................................................... 19 1.3.2 Imaging and Registration ................................................. 19 1.3.3 Results ............................................................................. 20 1.4 Imaging Protocols ....................................................................... 23 1.4.1 MR Imaging ..................................................................... 23 1.4.2 CT Imaging Protocol........................................................ 23 1.4.3 Bioluminescent Imaging ................................................... 24 1.4.4 BLT Reconstruction ......................................................... 24 vi 1.4.5 MR to BLT/CT Registration ........................................... 25 1.5 In Vivo Validation ...................................................................... 26 1.5.1 Overview and Approach ................................................... 26 1.5.2 Cell Culture and Transplantation .................................... 27 1.5.3 Animal Imaging Protocol ................................................. 27 1.5.4 Histological Analysis ........................................................ 28 1.5.5 Results ............................................................................. 28 1.6 Conclusions ................................................................................. 35 Chapter 2: Quantitative Bioluminescence Tomography using Prior Spatial Information ....................................................................................... 36 2.1 Motivation .................................................................................. 36 2.1.1 Spatial Prior Knowledge in BLT ...................................... 37 2.1.2 Approach in this Chapter ................................................. 38 2.2 Background Theory .................................................................... 40 2.2.1 Light Propagation in Biological Tissues ........................... 40 2.2.2 The Radiative Transfer Equation ..................................... 42 2.2.3 Solutions to the RTE ....................................................... 43 2.3 Finite Difference Method ............................................................ 46 2.3.1 Implementation ................................................................ 46 2.3.2 Boundary Conditions ....................................................... 47 2.3.3 Numerical Validation ....................................................... 48 2.3.4 Limitations of FDM Approach ......................................... 52 2.4 Finite Element Method ............................................................... 54 2.4.2 Implementation ................................................................ 54 2.4.3 Matrix Assembly .............................................................. 56 2.4.4 Conversion of Photon Density to Radiance ...................... 59 2.5 Implementation and Source Quantification ................................. 61 2.6 Method Validation .....................................................................