Marcus Wilhelmsson is an Associate Professor of Biophysical Chemistry at Chalmers University of Technology, Göteborg, Sweden.  He earned his Ph.D. in 2003 and thereafter entered the field of DNA-nanotechnology. In 2008 he joined Chalmers University of Technology as an Assistant Professor, where he is now working on the design, characterization and application of novel fluorescent nucleic acid base analogues.Yitzhak Tor is a Professor of Chemistry and Biochemistry and the George and Carol Lattimer Professor at the University of California, San Diego. He earned his Ph.D. in 1990 from the Weizmann Institute of Science and conducted postdoctoral work at the California Institute of Technology. His first faculty appointment was at the University of Chicago, followed by the University of California, San Diego since 1994.

"This book provides a thorough overview on the design and application of fluorescent analogs of biomolecular building blocks...The way the book is written makes reading enjoyable and relatively easy for readers who already have some knowledge on the subject as well as for beginners...Overall, the book is very well achieved, and I strongly recommend reading." (Angewandte Chemie International Edition May 2017)

• List of Contributors xvPreface xix1 Fluorescence Spectroscopy 1Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor1.1 Fundamentals of Fluorescence Spectroscopy 11.2 Common Fluorescence Spectroscopy Techniques 31.3 Summary and Perspective 10References 102 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15Renatus W. Sinkeldam and Yitzhak Tor2.1 Introduction 152.2 Naturally Occurring Emissive Biomolecular Building Blocks 162.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 182.4 Summary and Perspective 31References 323 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40Bo Albinsson and Bengt Nordén3.1 Transition Moments 403.2 Linear Dichroism 413.3 Magnetic Circular Dichroism 453.4 F̈orster Resonance Energy Transfer (FRET) 463.5 Fluorescence Anisotropy 473.6 Fluorescent Nucleobases 473.7 Fluorescent Peptide Chromophores 483.8 Site-Specific Linear Dichroism (SSLD) 503.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 503.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51References 534 Fluorescent Proteins: The Show Must go on! 55Gregor Jung4.1 Introduction 554.2 Historical Survey 554.3 Photophysical Properties 574.4 Photochemical Reactions 714.5 Ion Sensitivity 754.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 774.7 Prospects and Outlook 82Acknowledgments 82References 825 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91Patrick M. Durkin and Nediljko Budisa5.1 Introduction 915.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 975.3 Application of Fluorescent Building Blocks in Proteins 1115.4 Conclusions 1175.5 Prospects and Outlook 118Acknowledgments 120References 1206 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124Bruce A. Armitage6.1 Introduction 1246.2 Fluoromodule Development and Characterization 1266.3 Implementation 1326.4 Conclusions 1346.5 Prospects and Outlook 134Acknowledgments 134References 1347 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137Subhendu Sekhar Bag and Isao Saito7.1 Introduction 1377.2 Solvatochromic Fluorescent Nucleoside Analogs 1407.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 1417.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 1537.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 1547.6 Base-Selective Fluorescent ESF Probe 1637.7 Molecular Beacon (MB) and ESF Nucleosides 1677.8 Summary and Future Outlook 169Acknowledgments 170References 1708 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174Kirby Chicas and Robert H.E. Hudson8.1 Introduction 1748.2 Design and Characterization of Fluorescent C Analogs 1768.3 Implementation 1908.4 Conclusions 2028.5 Prospects and Outlook 202Acknowledgments 203References 2039 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine9.1 Introduction 2099.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 2099.3 Implementation 2159.4 Conclusions 2209.5 Prospects and Outlook 221References 22110 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224L. Marcus Wilhelmsson10.1 Introduction 22410.2 The Tricyclic Cytosine Family 22610.3 Development of the First Nucleic Acid Base Analog FRET Pair 23410.4 Conclusions 23810.5 Prospects and Outlook 238Acknowledgments 239References 23911 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke11.1 Introduction 24211.2 Design, Photophysical Properties, and Applications of Purine Mimics 24411.3 Implementation 25811.4 Conclusions 26511.5 Prospects and Outlook 265Appendix 268References 26812 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276Renatus W. Sinkeldam and Yitzhak Tor12.1 Introduction 27612.2 Designing Environmentally Sensitive Emissive Nucleosides 27912.3 Two Isomorphic Environmentally Sensitive Designs 28212.4 Probing Environmental Sensitivity 28312.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 29112.6 Summary 29312.7 Prospects and Outlook 294Acknowledgments 294References 29413 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297Michiko Kimoto, Rie Yamashige, and Ichiro Hirao13.1 Introduction 29713.2 Development of Unnatural Base Pair Systems and Their Applications 29913.3 Implementation 31013.4 Conclusions 31513.5 Prospects and Outlook 316Acknowledgments 317References 31714 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320Pete Crisalli and Eric T. Kool14.1 Introduction 32014.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 32214.3 Implementation of Fluorescent C-Glycoside Monomers 32714.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 33514.5 Implementation of Fluorescent C-Glycoside Oligomers 34214.6 Conclusions 35314.7 Prospects and Outlook 353Acknowledgments 354References 35415 Membrane Fluorescent Probes: Insights and Perspectives 356Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima ChaudhuriAbbreviations 35615.1 Introduction 35715.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 35815.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 35915.4 Pyrene: a Multiparameter Membrane Probe 36215.5 Conclusion and Future Perspectives 362Acknowledgments 364References 36416 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367Marek Cebecauer and Radek Šachl16.1 Introduction 36716.2 Lipids, Lipid Bilayers, and Biomembranes 36816.3 Lipid Phases, Phase Separation, and Lipid Ordering 37016.4 Fluorescent Probes for Membrane Studies 37016.5 Conclusions 38616.6 Prospects and Outlook 386Acknowledgments 386References 38717 Fluorescent Neurotransmitter Analogs 393James N. Wilson17.1 Introduction 39317.2 Design and Optical Properties of Fluorescent Neurotransmitters 39717.3 Applications of Fluorescent Neurotransmitters 40017.4 Conclusions 40417.5 Prospects and Outlook 405Acknowledgments 405References 406Index 409

Marcus Wilhelmsson is an Associate Professor of Biophysical Chemistry at Chalmers University of Technology, Göteborg, Sweden.  He earned his Ph.D. in 2003 and thereafter entered the field of DNA-nanotechnology. In 2008 he joined Chalmers University of Technology as an Assistant Professor, where he is now working on the design, characterization and application of novel fluorescent nucleic acid base analogues.Yitzhak Tor is a Professor of Chemistry and Biochemistry and the George and Carol Lattimer Professor at the University of California, San Diego. He earned his Ph.D. in 1990 from the Weizmann Institute of Science and conducted postdoctoral work at the California Institute of Technology. His first faculty appointment was at the University of Chicago, followed by the University of California, San Diego since 1994.

"This book provides a thorough overview on the design and application of fluorescent analogs of biomolecular building blocks...The way the book is written makes reading enjoyable and relatively easy for readers who already have some knowledge on the subject as well as for beginners...Overall, the book is very well achieved, and I strongly recommend reading." (Angewandte Chemie International Edition May 2017)

• List of Contributors xvPreface xix1 Fluorescence Spectroscopy 1Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor1.1 Fundamentals of Fluorescence Spectroscopy 11.2 Common Fluorescence Spectroscopy Techniques 31.3 Summary and Perspective 10References 102 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15Renatus W. Sinkeldam and Yitzhak Tor2.1 Introduction 152.2 Naturally Occurring Emissive Biomolecular Building Blocks 162.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 182.4 Summary and Perspective 31References 323 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40Bo Albinsson and Bengt Nordén3.1 Transition Moments 403.2 Linear Dichroism 413.3 Magnetic Circular Dichroism 453.4 F̈orster Resonance Energy Transfer (FRET) 463.5 Fluorescence Anisotropy 473.6 Fluorescent Nucleobases 473.7 Fluorescent Peptide Chromophores 483.8 Site-Specific Linear Dichroism (SSLD) 503.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 503.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51References 534 Fluorescent Proteins: The Show Must go on! 55Gregor Jung4.1 Introduction 554.2 Historical Survey 554.3 Photophysical Properties 574.4 Photochemical Reactions 714.5 Ion Sensitivity 754.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 774.7 Prospects and Outlook 82Acknowledgments 82References 825 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91Patrick M. Durkin and Nediljko Budisa5.1 Introduction 915.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 975.3 Application of Fluorescent Building Blocks in Proteins 1115.4 Conclusions 1175.5 Prospects and Outlook 118Acknowledgments 120References 1206 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124Bruce A. Armitage6.1 Introduction 1246.2 Fluoromodule Development and Characterization 1266.3 Implementation 1326.4 Conclusions 1346.5 Prospects and Outlook 134Acknowledgments 134References 1347 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137Subhendu Sekhar Bag and Isao Saito7.1 Introduction 1377.2 Solvatochromic Fluorescent Nucleoside Analogs 1407.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 1417.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 1537.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 1547.6 Base-Selective Fluorescent ESF Probe 1637.7 Molecular Beacon (MB) and ESF Nucleosides 1677.8 Summary and Future Outlook 169Acknowledgments 170References 1708 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174Kirby Chicas and Robert H.E. Hudson8.1 Introduction 1748.2 Design and Characterization of Fluorescent C Analogs 1768.3 Implementation 1908.4 Conclusions 2028.5 Prospects and Outlook 202Acknowledgments 203References 2039 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine9.1 Introduction 2099.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 2099.3 Implementation 2159.4 Conclusions 2209.5 Prospects and Outlook 221References 22110 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224L. Marcus Wilhelmsson10.1 Introduction 22410.2 The Tricyclic Cytosine Family 22610.3 Development of the First Nucleic Acid Base Analog FRET Pair 23410.4 Conclusions 23810.5 Prospects and Outlook 238Acknowledgments 239References 23911 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke11.1 Introduction 24211.2 Design, Photophysical Properties, and Applications of Purine Mimics 24411.3 Implementation 25811.4 Conclusions 26511.5 Prospects and Outlook 265Appendix 268References 26812 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276Renatus W. Sinkeldam and Yitzhak Tor12.1 Introduction 27612.2 Designing Environmentally Sensitive Emissive Nucleosides 27912.3 Two Isomorphic Environmentally Sensitive Designs 28212.4 Probing Environmental Sensitivity 28312.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 29112.6 Summary 29312.7 Prospects and Outlook 294Acknowledgments 294References 29413 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297Michiko Kimoto, Rie Yamashige, and Ichiro Hirao13.1 Introduction 29713.2 Development of Unnatural Base Pair Systems and Their Applications 29913.3 Implementation 31013.4 Conclusions 31513.5 Prospects and Outlook 316Acknowledgments 317References 31714 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320Pete Crisalli and Eric T. Kool14.1 Introduction 32014.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 32214.3 Implementation of Fluorescent C-Glycoside Monomers 32714.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 33514.5 Implementation of Fluorescent C-Glycoside Oligomers 34214.6 Conclusions 35314.7 Prospects and Outlook 353Acknowledgments 354References 35415 Membrane Fluorescent Probes: Insights and Perspectives 356Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima ChaudhuriAbbreviations 35615.1 Introduction 35715.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 35815.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 35915.4 Pyrene: a Multiparameter Membrane Probe 36215.5 Conclusion and Future Perspectives 362Acknowledgments 364References 36416 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367Marek Cebecauer and Radek Šachl16.1 Introduction 36716.2 Lipids, Lipid Bilayers, and Biomembranes 36816.3 Lipid Phases, Phase Separation, and Lipid Ordering 37016.4 Fluorescent Probes for Membrane Studies 37016.5 Conclusions 38616.6 Prospects and Outlook 386Acknowledgments 386References 38717 Fluorescent Neurotransmitter Analogs 393James N. Wilson17.1 Introduction 39317.2 Design and Optical Properties of Fluorescent Neurotransmitters 39717.3 Applications of Fluorescent Neurotransmitters 40017.4 Conclusions 40417.5 Prospects and Outlook 405Acknowledgments 405References 406Index 409

Marcus Wilhelmsson is an Associate Professor of Biophysical Chemistry at Chalmers University of Technology, Göteborg, Sweden.  He earned his Ph.D. in 2003 and thereafter entered the field of DNA-nanotechnology. In 2008 he joined Chalmers University of Technology as an Assistant Professor, where he is now working on the design, characterization and application of novel fluorescent nucleic acid base analogues.Yitzhak Tor is a Professor of Chemistry and Biochemistry and the George and Carol Lattimer Professor at the University of California, San Diego. He earned his Ph.D. in 1990 from the Weizmann Institute of Science and conducted postdoctoral work at the California Institute of Technology. His first faculty appointment was at the University of Chicago, followed by the University of California, San Diego since 1994.

"This book provides a thorough overview on the design and application of fluorescent analogs of biomolecular building blocks...The way the book is written makes reading enjoyable and relatively easy for readers who already have some knowledge on the subject as well as for beginners...Overall, the book is very well achieved, and I strongly recommend reading." (Angewandte Chemie International Edition May 2017)

• List of Contributors xvPreface xix1 Fluorescence Spectroscopy 1Renatus W. Sinkeldam, L. Marcus Wilhelmsson, and Yitzhak Tor1.1 Fundamentals of Fluorescence Spectroscopy 11.2 Common Fluorescence Spectroscopy Techniques 31.3 Summary and Perspective 10References 102 Naturally Occurring and Synthetic Fluorescent Biomolecular Building Blocks 15Renatus W. Sinkeldam and Yitzhak Tor2.1 Introduction 152.2 Naturally Occurring Emissive Biomolecular Building Blocks 162.3 Synthetic Fluorescent Analogs of Biomolecular Building Blocks 182.4 Summary and Perspective 31References 323 Polarized Spectroscopy with Fluorescent Biomolecular Building Blocks 40Bo Albinsson and Bengt Nordén3.1 Transition Moments 403.2 Linear Dichroism 413.3 Magnetic Circular Dichroism 453.4 F̈orster Resonance Energy Transfer (FRET) 463.5 Fluorescence Anisotropy 473.6 Fluorescent Nucleobases 473.7 Fluorescent Peptide Chromophores 483.8 Site-Specific Linear Dichroism (SSLD) 503.9 Single-Molecule Fluorescence Resonance Energy Transfer (smFRET) 503.10 Single-Molecule Fluorescence-Detected Linear Dichroism (smFLD) 51References 534 Fluorescent Proteins: The Show Must go on! 55Gregor Jung4.1 Introduction 554.2 Historical Survey 554.3 Photophysical Properties 574.4 Photochemical Reactions 714.5 Ion Sensitivity 754.6 Relation Microscopy–Spectroscopy for Fluorescent Proteins 774.7 Prospects and Outlook 82Acknowledgments 82References 825 Design and Application of Autofluorescent Proteins by Biological Incorporation of Intrinsically Fluorescent Noncanonical Amino Acids 91Patrick M. Durkin and Nediljko Budisa5.1 Introduction 915.2 Design and Synthesis of Fluorescent Building Blocks in Proteins 975.3 Application of Fluorescent Building Blocks in Proteins 1115.4 Conclusions 1175.5 Prospects and Outlook 118Acknowledgments 120References 1206 Fluoromodules: Fluorescent Dye–Protein Complexes for Genetically Encodable Labels 124Bruce A. Armitage6.1 Introduction 1246.2 Fluoromodule Development and Characterization 1266.3 Implementation 1326.4 Conclusions 1346.5 Prospects and Outlook 134Acknowledgments 134References 1347 Design of Environmentally Sensitive Fluorescent Nucleosides and their Applications 137Subhendu Sekhar Bag and Isao Saito7.1 Introduction 1377.2 Solvatochromic Fluorescent Nucleoside Analogs 1407.3 Fluorescently Labeled Nucleosides and Oligonucleotide Probes: Covalent Attachment of Solvatochromic Fluorophores Onto the Natural Bases 1417.4 Nucleosides with Dual Fluorescence for Monitoring DNA Hybridization 1537.5 Approach for Developing Environmentally Sensitive Fluorescent (ESF) Nucleosides 1547.6 Base-Selective Fluorescent ESF Probe 1637.7 Molecular Beacon (MB) and ESF Nucleosides 1677.8 Summary and Future Outlook 169Acknowledgments 170References 1708 Expanding The Nucleic Acid Chemist’s Toolbox: Fluorescent Cytidine Analogs 174Kirby Chicas and Robert H.E. Hudson8.1 Introduction 1748.2 Design and Characterization of Fluorescent C Analogs 1768.3 Implementation 1908.4 Conclusions 2028.5 Prospects and Outlook 202Acknowledgments 203References 2039 Synthesis and Fluorescence Properties of Nucleosides with Pyrimidopyrimidine-Type Base Moieties 208Kohji Seio, Takashi Kanamori, Akihiro Ohkubo, and Mitsuo Sekine9.1 Introduction 2099.2 Discovery, Design, and Synthesis of Pyrimidopyrimidine Nucleosides 2099.3 Implementation 2159.4 Conclusions 2209.5 Prospects and Outlook 221References 22110 Förster Resonance Energy Transfer (FRET) Between Nucleobase Analogues – a Tool for Detailed Structure and Dynamics Investigations 224L. Marcus Wilhelmsson10.1 Introduction 22410.2 The Tricyclic Cytosine Family 22610.3 Development of the First Nucleic Acid Base Analog FRET Pair 23410.4 Conclusions 23810.5 Prospects and Outlook 238Acknowledgments 239References 23911 Fluorescent Purine Analogs that Shed Light on DNA Structure and Function 242Anaëlle Dumas, Guillaume Mata, and Nathan W. Luedtke11.1 Introduction 24211.2 Design, Photophysical Properties, and Applications of Purine Mimics 24411.3 Implementation 25811.4 Conclusions 26511.5 Prospects and Outlook 265Appendix 268References 26812 Design and Photophysics of Environmentally Sensitive Isomorphic Fluorescent Nucleosides 276Renatus W. Sinkeldam and Yitzhak Tor12.1 Introduction 27612.2 Designing Environmentally Sensitive Emissive Nucleosides 27912.3 Two Isomorphic Environmentally Sensitive Designs 28212.4 Probing Environmental Sensitivity 28312.5 Recent Advancements in Isomorphic Fluorescent Nucleoside Analogs 29112.6 Summary 29312.7 Prospects and Outlook 294Acknowledgments 294References 29413 Site-Specific Fluorescent Labeling of Nucleic Acids by Genetic Alphabet Expansion Using Unnatural Base Pair Systems 297Michiko Kimoto, Rie Yamashige, and Ichiro Hirao13.1 Introduction 29713.2 Development of Unnatural Base Pair Systems and Their Applications 29913.3 Implementation 31013.4 Conclusions 31513.5 Prospects and Outlook 316Acknowledgments 317References 31714 Fluorescent C-Nucleosides and their Oligomeric Assemblies 320Pete Crisalli and Eric T. Kool14.1 Introduction 32014.2 Design, Synthesis, Characterization, and Properties of Fluorescent C-Glycoside Monomers 32214.3 Implementation of Fluorescent C-Glycoside Monomers 32714.4 Oligomers of Fluorescent C-Glycosides: Design, Synthesis, and Properties 33514.5 Implementation of Fluorescent C-Glycoside Oligomers 34214.6 Conclusions 35314.7 Prospects and Outlook 353Acknowledgments 354References 35415 Membrane Fluorescent Probes: Insights and Perspectives 356Amitabha Chattopadhyay, Sandeep Shrivastava, and Arunima ChaudhuriAbbreviations 35615.1 Introduction 35715.2 NBD-Labeled Lipids: Monitoring Slow Solvent Relaxation in Membranes 35815.3 n-AS Membrane Probes: Depth-Dependent Solvent Relaxation as Membrane Dipstick 35915.4 Pyrene: a Multiparameter Membrane Probe 36215.5 Conclusion and Future Perspectives 362Acknowledgments 364References 36416 Lipophilic Fluorescent Probes: Guides to the Complexity of Lipid Membranes 367Marek Cebecauer and Radek Šachl16.1 Introduction 36716.2 Lipids, Lipid Bilayers, and Biomembranes 36816.3 Lipid Phases, Phase Separation, and Lipid Ordering 37016.4 Fluorescent Probes for Membrane Studies 37016.5 Conclusions 38616.6 Prospects and Outlook 386Acknowledgments 386References 38717 Fluorescent Neurotransmitter Analogs 393James N. Wilson17.1 Introduction 39317.2 Design and Optical Properties of Fluorescent Neurotransmitters 39717.3 Applications of Fluorescent Neurotransmitters 40017.4 Conclusions 40417.5 Prospects and Outlook 405Acknowledgments 405References 406Index 409