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Title: Nonlinear Optics of Fluorescent Proteins: Characterization and Application in Second-Harmonic Generation (SHG) Microscopy.
Other Titles: Niet-Lineaire Optica van Fluorescente Proteïnen: Karakterisatie en Toepassing in Tweede-Harmonische Generatie (SHG) Microscopie.
Authors: De Meulenaere, Evelien
Issue Date: 25-May-2012
Abstract: This project aimed at investigating the applicability of fluorescent proteins as probes in second-harmonic generation (SHG) imaging in order to obtain additional intermolecular structural information from already existing, and working cellular systems expressing fusion constructs of proteins of interest covalently linked to fluorescent proteins. SHG microscopy even has the potential to visualize conformational changes in vivo in certain cases.During this project, the second-order nonlinear optical properties of nine fluorescent proteins, measured by the first hyperpolarizability, ß, were determined by frequency-resolved hyper-Rayleigh scattering at the fundamental wavelength (first harmonic) of 800 nm. The chromophores of the fluorescent proteins were confirmed to exhibit SHG, and may be used for optical techniques based on SHG. The intensity of their response depends not only on the wavelength used, due to resonance enhancement, but also on the size of the conjugated system, forming the chromophore, of the proteins. A stronger response, correlated with a larger ß, is inherently linked to a longer conjugated system, also associated with a red-shift in the absorption and fluorescence spectra. This counts for all measured proteins, as long as the chromophore has no inversion centers, which is not the case for the enhanced yellow fluorescent protein, eYFP.SHG microscopy was successfully performed on cultured cells stained with dyes especially designed for nonlinear imaging. Fluorescent proteins turn out to be weak in comparison to these samples for several reasons. Firstly, the measured ߒs are considerably smaller than those of the small organic dyes; secondly, the concentration of the fluorescent proteins by expression in the cells will usually be remarkably lower; and thirdly, the fluorescent proteins are linked to proteins-of-interest by a relatively flexible linker, compromising the level of order in the intermolecular orientation of the proteins, an important condition for SHG.However, this project also indicates possible solutions to these downsides, as the ߒs of the most red-shifted proteins are approaching the ߒs of the dyes. More red-shifted proteins are already available and can be tested using higher wavelengths, increasing resonance enhancement with the same effort. Also, the effect of non-natural amino acids can be investigated, as well as methods to increase control over the orientation of the chromophores. When these directions are followed, SHG microscopy may prove effective with fluorescent proteins.
Table of Contents: TABLE OF CONTENTS I
SUMMARY III
SAMENVATTING IV
LIST OF ABBREVIATIONS V
LIST OF SYMBOLS VII
LIST OF NATURAL AMINO ACIDS (BUILDING BLOCKS FOR PROTEINS) VIII
NUCLEOTIDES (BUILDING BLOCKS FOR DNA) VIII
CURRICULUM VITAE IX
CHAPTER 1: INTRODUCTION 1
SCOPE 2
SHORT HISTORY 3
FLUORESCENT PROTEINS 3
NONLINEAR OPTICS 5
SECOND-HARMONIC GENERATION IN BIOMEDICAL RESEARCH 12
THESIS GUIDE 18
REFERENCES 21
CHAPTER 2: SECOND-ORDER NONLINEAR OPTICAL PROPERTIES OF FLUORESCENT PROTEINS FOR SECOND-HARMONIC IMAGING 23
INTRODUCTION 25
MATERIALS AND METHODS 26
RESULTS 29
DISCUSSION 33
CONCLUSIONS AND PERSPECTIVES 35
ACKNOWLEDGEMENTS 35
REFERENCES 36
CHAPTER 3: NONLINEAR OPTICAL PROPERTIES OF MSTRAWBERRY AND MCHERRY FOR SECOND HARMONIC IMAGING 39
INTRODUCTION 41
MATERIALS AND METHODS 43
RESULTS 45
DISCUSSION 48
CONCLUSIONS AND PERSPECTIVES 50
ACKNOWLEDGEMENTS 50
REFERENCES 51
CHAPTER 4: BIO-INSPIRED NANO-ENGINEERING AND GENETIC MODIFICATION FOR NONLINEAR OPTICAL IMAGING53
ABSTRACT 54
1 INTRODUCTION 54
2 MATERIALS AND METHODS 56
3 RESULTS 58
4 PERSPECTIVES 61
5 CONCLUSIONS 62
6 REFERENCES 62
REMARKS: 65
CHAPTER 5: PREDICTION OF FIRST HYPERPOLARIZABILITY OF FLUORESCENT PROTEINS 67
INTRODUCTION 68
METHODS 69
RESULTS 70
DISCUSSION 71
CONCLUSIONS 73
ACKNOWLEDGEMENTS 74
REFERENCES 74
CHAPTER 6: A NEW YELLOW FLUORESCENT PROTEIN FOR SECOND-HARMONIC IMAGING 75
INTRODUCTION 76
MATERIALS AND METHODS 78
RESULTS 82
DISCUSSION 86
CONCLUSIONS 87
REFERENCES 88
CHAPTER 7: SIMULTANEOUS SHG AND 2PEF IMAGING USING A NEW TYPE OF SELECTIVE MARKERS 91
ABSTRACT 92
INTRODUCTION 92
MATERIALS AND METHODS 94
RESULTS 96
DISCUSSION 101
CONCLUSIONS 102
ACKNOWLEDGEMENTS 102
REFERENCES 103
CHAPTER 8: MOLECULAR ENGINEERING OF CHROMOPHORES FOR COMBINED SECOND-HARMONIC AND TWO-PHOTON FLUORESCENCE IN CELLULAR IMAGING 105
A INTRODUCTION 107
B MATERIALS AND METHODS 109
C RESULTS 113
D DISCUSSION 119
E CONCLUSIONS 125
F NOTES AND REFERENCES 127
COLOCALIZATION EXPERIMENT 130
CHAPTER 9: PROBING LIVE SAMPLES IN SECOND-HARMONIC GENERATION MICROSCOPY USING SPECIFIC MARKERS AND FLUORESCENT PROTEINS 135
ABSTRACT 136
INTRODUCTION 137
MATERIALS AND METHODS 139
RESULTS 141
DISCUSSION 144
CONCLUSIONS AND PERSPECTIVES 146
REFERENCES 147
BROADER CONTEXT OF THE EXPERIMENT 149
CHAPTER 10: NON-NATURAL AMINO ACIDS: A NEW AREA OF RESEARCH FOR SECOND-HARMONIC IMAGING OF PROTEINS? 153
INTRODUCTION 154
METHODS 155
RESULTS AND DISCUSSION 155
CONCLUSIONS AND PERSPECTIVES 159
REFERENCES 160
CHAPTER 11: GENERAL CONCLUSIONS AND PERSPECTIVES 161
APPENDICES 169
APPENDIX A SAMPLE PREPARATION 170
APPENDIX B DATA ANALYSIS HRS 178
APPENDIX C OPTIMIZATION OF CULTURE CONDITIONS 182
APPENDIX D PRIMERS
ISBN: 978-90-8826-245-6
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre of Microbial and Plant Genetics
Molecular Imaging and Photonics

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