Development of a spectrally resolved fluorescence 3D for lifetime imaging microscopy precision measurements in the nanometer range [Elektronische Ressource] / vorgelegt von John Wallis

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INAUGURAL-DISSERTATION Zur Erlangung der Doktorwürde Der Naturwissenschaftlich-Mathematischen Gesamtfakultät Der Ruprecht-Karls-Universität Heidelberg Vorgelegt von Diplom-Mathematiker John Wallis Aus Aurora, Colorado Tag der Mündlichen Prüfung: 11. Juli 2003 DEVELOPMENT OF A SPECTRALLY-RESOLVED FLUORESCENCE 3D FOR LIFETIME IMAGING MICROSCROPY PRECISION MEASUREMENTS IN THE NANOMETER RANGE Gutachter: Prof. Dr. Jürgen Wolfrum Prof. Dr. Joachim Pius SpatzTo my wife ii ACKNOWLEDGMENTS I would like to thank my wife for her endless moral support during this work. I would also like to thank my parents, my wife’s parents, and my sons, Charles and Philip. I would also like to thank all the people at the chemical and numerical sections of the University of Heidelberg for their support. iii SUMMARY This work developed a program to monitor and collect florescence photons from single molecules in three-dimensional domains with high precision and developed tools for viewing such data. The resulting scanned images reveals better understanding of the interplay between the molecules. These images also help the researcher find areas of interest and he can quickly measure in more detail these areas.

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Published 01 January 2003
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INAUGURAL-DISSERTATION Zur Erlangung der Doktorwürde Der Naturwissenschaftlich-Mathematischen Gesamtfakultät Der Ruprecht-Karls-Universität Heidelberg         
Vorgelegt von Diplom-Mathematiker John Wallis Aus Aurora, Colorado     
Tag der Mündlichen Prüfung: 11. Juli 2003    
 Gutachter: Prof. Dr. Jürgen Wolfrum                                 Prof. Dr. Joachim Pius Spatz                                   
OLEVEDFO TNEMPDEF OSVLSEECULROPECT A SY-RERALLG INCRMIE IMAGIML ROTEFI ECNF D3EMENTS IN MEASURRPCESIOISORCPO Y          E NGRA RETEMONAN EHT N                      
To my wife
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ACKNOWLEDGMENTS 
I would like to thank my wife for her endless moral support during this work. I would
also like to thank my parents, my wife’s parents, and my sons, Charles and Philip. I
would also like to thank all the people at the chemical and numerical sections of the
University of Heidelberg for their support.
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SUMMARY
This work developed a program to monitor and collect florescence photons from single molecules in three-dimensional domains with high precision and developed tools for viewing such data. The resulting scanned images reveals better understanding of the interplay between the molecules. These images also help the researcher find areas of interest and he can quickly measure in more detail these areas. This program was written with Microsoft's Visual C that interfaced with a Queensgate NPS3330 nanopositioning table and an SPC-630 time correlated single-photon detection card. Micrometer volumes can be scanned using confocal microscopy techniques during which single molecules tagged with fluorescent dyes are excited by a pulsed laser. In a few nanoseconds the dyes will fluoresce. Several (100-1000) photons will move in the direction of one or more avalanche diode detectors (APD) that send signals to the photon detection card. The SPC card receives and orders incoming photons and sums them forming a decay (lifetime profile) curve. The SPC-630 card has a configurable memory capable of storing many decay curves of short durations, usually in microseconds. Blocks of these curves are periodically saved to disk for post-analysis. In the post-analysis, the program enables the user to choose which channels he wishes to use for lifetime estimations. Minimum/maximum boundary values can be given for a given variable for filtering permanent information. The user can also use previous scans to zoom in on certain domain areas. Information pertaining to selected individual pixels can be also collected and exported to other programs, such as Excel. This work developed a method for the 3D imaging of cells using spectrally – resolved fluorescence lifetime imaging microscopy (SFLIM)
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TABLE OF CONTENTS
ACKNOWLEDGMENTS ..............................................................................................III SUMMARY......................................................................................................................IV LIST OF TABLES ..........................................................................................................VI LIST OF FIGURES ...................................................................................................... VII 1. INTRODUCTION AND MOTIVATION ................................................................... 1 2. DESCRIPTION ........................................................................................................... 11 3. MATERIALS AND METHODS................................................................................ 13 3.1. QUEENSGATE NPS3330 .............................................................................. 14 3.2. SPC 630 ............................................................................................................ 17 3.3. THE PROGRAM ............................................................................................ 22 3.3.1. The Scanning....................................................................... 22 3.3.2. Postprocessing ..................................................................... 27 3.3.3. Miscellaneous Dialogs ........................................................ 31 3.3.3. The Visualization ................................................................ 33 4. EXAMPLES................................................................................................................. 34 4.1. A DEAD CELL................................................................................................ 34 4.2. BREAST CANCER......................................................................................... 36 5. DISCUSSION AND OUTLOOK ............................................................................... 37 APPENDICES ................................................................................................................. 40 A.  ................................................................... 40INDEX OF ABBREVIATIONS B. THE SCANNING ROUTINES .................................................................... 42 C.  ............................................................... 48THE SCANNED FILE FORMAT D. PROGRAM FILES ....................................................................................... 49 Primary files .................................................................................. 49 Secondary files .............................................................................. 50 BIBLIOGRAPHY ........................................................................................................... 51 
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