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Using Optical Imaging Methods to Assess Laser-Tissue Interactions USING OPTICAL IMAGING METHODS TO ASSESS LASER-TISSUE INTERACTIONS By Gerald Joseph Wilmink Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Engineering December, 2007 Nashville, Tennessee Approved: Professor Eric Duco Jansen Professor Anita Mahadevan-Jansen Professor Jeffrey M. Davidson Professor Lillian B. Nanney Professor Susan R. Opalenik Professor Frederick R. Haselton Copyright © 2007 by Gerald Joseph Wilmink All Rights Reserved ACKNOWLEDGEMENTS I thank God for the gift of life and for all of the wonderful service opportunities that He has made available to me. I am very grateful for my supportive mother, my dad, Joe Gambino, my grandparents, and my lovely wife Kimberly. Without such a wonderful support system this dissertation would not have been possible. I am also grateful for my wonderful adviser, Dr. Eric Duco Jansen. For without his patience and support I would not be where I am today. I will never forget the trips we took together to ASLMS and the Jansen summer lab parties. I am also grateful for Dr. Jansen and Anita Mahadevan- Jansen for paving the way for my next research opportunity down in San Antonio. I feel honored to have the chance to work with Dr. Pat Roach, and I look forward to having the opportunity to use my laser skills in the area of Terahertz radiation research. I am also very grateful for each member of my Ph.D. committee. I doubt I will ever meet anyone quite like Dr. Susan Opalenik. Dr. Opalenik was always there for me with an encouraging word and an open ear. During my most difficult times she was like a stream in the desert. I feel blessed for the opportunity to work with Dr. Jeff Davidson. He is one of the most brilliant scientists that I have ever met and I feel honored that I had the chance to work with him. I will never forget the many fruitful discussions that Dr. Davidson, Dr. Jansen, and I shared together in the BME conference room. I also really enjoyed working with Dr. Lillian Nanney. She taught me so much about immunohistochemistry, and she always made time available for me to use her microscope (at any hour of the day!!!). For without that microscope I would not have the beautiful histological images that are in this dissertation, and I would also have a healthy set of working retinas. I am also very grateful for time that I shared with Dr. Frederick Haselton. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .................................................................................................iii LIST OF TABLES............................................................................................................viii LIST OF FIGURES........................................................................................................... ix LIST OF SYMBOLS ......................................................................................................... xi Chapter Page I. INTRODUCTION...................................................................................................... 1 1.1. Introduction ..................................................................................................... 2 1.2. Specific Aims...................................................................................................4 1.3. Dissertation Outline......................................................................................... 7 1.4. Works Cited.....................................................................................................8 II. BACKGROUND AND SIGNIFICANCE .................................................................... 9 2.1. Introduction ...................................................................................................10 2.2. Properties of skin .......................................................................................... 11 2.2.1. Skin composition and morphology ................................................... 11 2.2.2. Mechanical properties ....................................................................... 13 2.3. Laser energy deposition and transport.......................................................... 14 2.3.1. Laser background ............................................................................ 14 2.3.2. Laser optical properties: absorption and scattering........................... 16 2.3.3. Photothermal effects ......................................................................... 19 2.3.4. Measuring tissue temperatures......................................................... 22 2.3.5. Thermal history ................................................................................. 25 2.4. Dermatologic laser applications .................................................................... 28 2.4.1. Aesthetic procedures......................................................................... 28 2.4.2. Surgical ablation procedures............................................................. 29 2.5. Heat shock proteins ..................................................................................... 31 2.6. Wound healing .............................................................................................. 35 2.6.1. Wound healing response .................................................................. 35 2.6.2. Perfusion ...........................................................................................38 2.6.3. Enhancing wound repair ................................................................... 39 2.7. Tissue preconditioning .................................................................................. 41 2.7.1. Preconditioning mechanism .............................................................. 41 2.7.2. Laser preconditioning and wound repair ........................................... 42 2.8. Wound repair skin models............................................................................ 45 2.9. Molecular and optical imaging...................................................................... 49 2.10. Works cited .................................................................................................. 56 iv III. ASSESSING LASER-TISSUE DAMAGE WITH BIOLUMINESCENT IMAGING ... 63 3.1. Abstract ......................................................................................................... 64 3.2. Introduction.................................................................................................... 65 3.3. Materials and methods .................................................................................. 68 3.3.1. Cell culture conditions ...................................................................... 68 3.3.2. Recombinant adenovirus construction ............................................. 69 3.3.3. Virus propagation and purification.................................................... 69 3.3.4. Construction of organotypic raft cultures.......................................... 70 3.3.5. Water bath and laser irradiation experiments................................... 70 3.3.6. Laser radiation with carbon dioxide laser ......................................... 71 3.3.7. Bioluminescent imaging ................................................................... 72 3.3.8. Bioluminescent imaging system....................................................... 72 3.3.9. Bioluminescent imaging of cell cultures ........................................... 73 3.3.10. Bioluminescent imaging of laser-treated rafts .................................. 73 3.3.11. ELISA assays................................................................................... 74 3.3.12. RNA preparation and Real-time RT-PCR......................................... 74 3.3.13. Flow cytometry ................................................................................. 75 3.3.14. Histology and immunohistochemistry............................................... 76 3.3.15. Bioluminescent microscopy.............................................................. 76 3.3.16. In situ transfections with adenovirus ................................................ 77 3.3.17. Co-localizing luciferase expression to hsp70 expression................. 77 3.4. Results .......................................................................................................... 78 3.4.1. Schematic representation of experimental methodology .................. 78 3.4.2. Bioluminescent intensity of hsp70 driven luciferase in NHDFs ......... 79 3.4.3. Transcriptional and translational profiles for skin cells...................... 80 3.4.4. Transfection is cell type dependent................................................... 83 3.4.5. Transient transfections...................................................................... 85 3.4.6. Homogenously transfecting rafts in situ ........................................... 86 3.4.7. Bioluminescence of laser irradiated raft samples.............................. 88 3.4.8. Co-localization of hsp70 on laser irradiated rafts .............................. 88 3.5. Discussion ..................................................................................................... 91 3.5.1. Characterizing the hsp70 response................................................... 92 3.5.2. Transient transfections ...................................................................... 95 3.5.3. Cell type dependent hsp70 expression ............................................. 96 3.5.4. Efficacy and
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