Nonlinear Optics of Fluorescent Proteins: Characterization and Application in Second-Harmonic Generation (SHG) Microscopy.

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 ß s 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 ß s of the most red-shifted proteins are approaching the ß s 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 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 PRIMERSnrpages: 203status: publishe