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In Vivo Confocal Microscopy in Turbid Media In Vivo Confocal Microscopy In Turbid Media Daniel S. Gareau B.S., Electrical Engineering, University of Vermont, Burlington, Vermont (1999) M.S., Electrical Engineering, Oregon Graduate Institute of Science & Technology, Beaverton, Oregon (2001) A thesis presented jointly to faculty of the OGI School of Science & Engineering at Oregon Health & Science University in partial fulfillment of the requirements for the degree Doctor of Philosophy in Biomedical Engineering. December 2006 © 2006 Dan Gareau ii The dissertation “Confocal Microscopy in Turbid Media and mice” by Daniel Gareau has been examined and approved by the following Examination Committee: Steven L. Jacques Professor Thesis Research Advisor Scott A. Prahl Assistant Professor Sean J. Kirkpatrick Associate Professor Molly Kulesz-Martin Professor iii Acknowledgments Sometimes, when I see how awesomely beautiful and insanely robust nature is I just want to give up. Who are we humans (victims of seeing only what we want to see) to even attempt to understand the natural world and ourselves? Then I remember that it’s already inside us and quite literally composes every fiber of our being. The time for “otherization” is over. The outward quest for the universe is only matched by the inward quest for the essence of our being. This quest requires tools like microscopes. Observation of the intense microscopic complexity of living tissue with optical microscopes requires building blocks such as knowing how light acts and interacts. This bottom-up approach, which starts with known properties of the building blocks and works toward microscope synthesis, is complemented by the top-down approach of biology which starts with the organism and work toward the properties of the building blocks. Engineering has traditionally taken a bottom-up approach. For example, combining the functional properties of the wheel and combustion engine one can synthesize a car, or filling a resonating cavity with a population-inverted optical gain medium, one can synthesize a LASER (Light Amplification by the Stimulated Emission of Radiation). The bottom-up approach is limited though. The problem (as pointed out to me by Dr. Anna Devor) with bottom-up methodology is that in biology, every time one thinks one knows the properties of a building block, the rug tends to get pulled out with the discovery that the building block is actually composed of smaller blocks which themselves are composed of yet smaller blocks, et. cetera. Although we all know that the truth is out there, getting to that truth is a long race and (I hope) the work here is just a few strides in that direction. The rigorous analysis probably lies in the use of fractal geometry, but for those of us who aren’t there yet (because fractal geometry is challenging), the solution is to pick a iv domain on nature’s infinite tree and solve both the forward and reverse problems simultaneously. This is biomedical engineering since biomedical engineers must be good top-down thinkers in order to analyze and good bottom-up thinkers in order to synthesize. This thesis involves the development of a tool for microscopic imaging capable of elucidating the structure and function of living cells, the building blocks of our bodies. It represents a small step on an immense journey. Live-tissue imaging is essential to understand life because it reveals the form of building blocks in the context of their function. Unlike noninvasive imaging techniques like x-ray or MRI technologies, light microscopy in the visible spectrum provides contrast that we can all relate to and understand. In a way, this is the most profound domain because it’s all around us and intimately connected with our perception and lives. I’d like to thank my family for putting me up to the plate, my advisor for helping me hit the ball, and my committee for playing the field. I’d like to thank my friends/colleagues in academia: Kirstin Engelking, Ted Moffitt, Yin-Chu Chen, Jessica Remella-Roman, Paulo Bargo and John Adams. I’d like to give a special thanks to Steve Jacques, and Scott Prahl who are two of the greatest teachers/people on the planet and taught me how thinking harder is cool. v Table of Contents Aknowledgements…………………………………………………………iv Table of Contents…………………………………………………………vi List of Tables………………………………………………………………x List of Figures……………………………………………………………..xi Abstract………………………………………………………………….xvii Chapter 1 - Introduction - 1.A Confocal Scanning Laser Microscopy - 1.A.1 The Confocal Principal................................................................1 - 1.A.2 Fluorescence..................................................................................4 - 1.A.3 Multifocal Scanning…………………………………………….5 - 1.B Confocal Microscopy in Turbid, Living Tissue - 1.B.1 The Point Spread Function (PSF)……………………………...6 - 1.B.2 Reflectance Confocal Microscopy……………………………...9 - 1.B.3 Comparable Imaging Techniques…………………………….10 - 1.B.4 In Vivo Fluorescence Confocal Microscopy…………………..10 - 1.B.5 Outline of Chapters………………………………………........14 Part I: Basic Science Studies Chapter 2 - Instrumentation - 2.A Overview and Component List - 2.A.1 System Layout……………………………………………........15 - 2.A.2 Three Channels…………………………………………..........21 - 2.B Optics - 2.B.1 Filters…………………………………………………………...22 - 2.B.2 Scanning……………………………………………………….24 - 2.B.3 Magnification……………………………………………….....25 - 2.B.4 Pinhole/Ring Detector…………………………………………29 - 2.C Electronics - 2.C.1 Scanning………………………………………………………..30 - 2.C.2 Detection…………………………………………………..........31 - 2.C.3 Data Acquisition and Processing……………………………..33 - 2.C.4 Operating Instructions………………………………………..39 Chapter 3 - Imaging in Non-Scattering Media - 3.A Reflectance Mode Calibration - 3.A.1 Axial Point-Spread Function for rCSLM…………………….42 - 3.A.2 Reflectance Normalization…………………………………….44 vi - 3.A.3 Field of View Calibration………………………………...........52 - 3.B Fluorescence mode calibration - 3.B.1 Microsphere Images in Non-scattering Medium……………56 - 3.B.2 Imaging a Fluorescent Neuron………………….....................59 - 3.B.3 Imaging the Iris of a Living Mouse…………………………..62 - 3.C Conclusions……………………………………………………………...63 Chapter 4 - Novel Confocal Detection for Imaging in Scattering Media - 4.A Introduction……………………………………………………………...65 - 4.A.1 The Effect of Scattering on in vivo Confocal Microscopy……68 - 4.A.2 Theoretical Example Monte Carlo Point-Spread Function…72 - 4.A.3 Analysis…………………………………………………………74 - 4.B Modified Confocal Detection…………………………………………….76 - 4.C Microsphere Imaging………………………………………………….....79 Chapter 5 - Monte Carlo Simulation - 5.A Introduction………………………………………………………………88 - 5.B Photon Launch - 5.B.1 Gaussian Beam at the Tissue Surface………………………...88 - 5.B.2 Focusing to an Airy Distribution……………………………...89 - 5.C Photon Propagation - 5.C.1 Propagation in Media………………………………………….95 - 5.C.2 Fluorescence……………………………………………………96 - 5.C.3 Confocal Detection……………………………….....................98 - 5.D Discussion……………………………………………………………….101 vii Part II: Mouse Studies Chapter 6 - Reflectance Mode Confocal Microscopy of Multiple Tissue Types - 6.A Abstract…………………………………………………………………102 - 6.B Introduction…………………………………………………………….102 - 6.C Materials and Methods 6.C.1 Gel with Polystyrene Microspheres…………………………..103 6.C.2 Monte Carlo Simulation as Numerical Experiment…………105 6.C.3 Mouse Tissue Studies………………………….………………105 - 6.D Analysis Grid…………………………………………………………...106 - 6.E Results 6.E.1 Analysis Grid Using Eq. 6.2…………………………………..109 6.E.2 Analysis Grid Using Monte Carlo Simulation……………….110 6.E.3 Mouse Tissue Studies………………………………………….112 - 6.F Discussion…………………………………………………………….....119 Chapter 7 - Noninvasive imaging of melanoma with reflectance mode confocal scanning laser microscopy in a murine model. - 7.A Abstract…………………………………………………………………120 - 7.B Introduction……………………………………………….....................120 - 7.C Materials and methods - 7.C.1 Animals………………………………………………………..123 - 7.C.2 rCSLM………………………………………………………...125 - 7.C.3 Experimental Protocol………………………………………..126 - 7.D Results - 7.D.1 Histopathology………………………………………………..128 - 7.D.2 rCSLM……….……………………………………………......129 - 7.E Discussion……………………………………………………………….140 Chapter 8 - Imaging Melanoma in a Murine Model using Reflectance- mode Confocal Scanning Laser Microscopy and Polarized Light Imaging. - 8.A Abstract…………………………………………………………………142 - 8.B Introduction……………………………………………….....................143 - 8.C Materials and Methods - 8.C.1 Animals………………………………………………………..145 - 8.C.2 rCSLM.………………………………………………………..146 - 8.C.3 Polarized Light Imaging…………………..............................147 - 8.D Results - 8.D.1 rCSLM.……………………………………………………….151 - 8.D.2 Polarized Light Imaging…………………..............................156 - 8.E Discussion………………………………………………….....................159 viii Chapter 9 - Optical Properties of Murine Skin at 488 nm - 9.A Abstract………………………………………………………………….161 - 9.B Introduction……………………………………………………………..161 - 9.C Materials and methods…………………………………………………162 - 9.D Analysis………………………………………………………………….163 - 9.E Results…………………………………………………………………...169 - 9.F Discussion………………………………………………………………..172 Chapter 10 - Confocal Fluorescence Spectroscopy of Subcutaneous Cartilage Expressing Green Fluorescent Protein versus Cutaneous Collagen Autofluorescence. - 10.A Abstract………………………………………………………………..174 - 10.B Introduction…………………………………………………………...175 - 10.C Materials and Methods - 10.C.1 Animal Model……………………………………………….177 - 10.C.2 Confocal System…………………………………………….177 - 10.C.3 Optical Fiber Probe…………………………………………179 - 10.C.4 Whole Animal Experiment…………………………………179 -
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