Fluorescence reporter is the key element of any sensing or imaging technology. Its optimal choice and implementation is very important for increasing the sensitivity, precision, multiplexing power, and also the spectral, temporal, and spatial reso- tion in different methods of research and practical analysis. Therefore, design of ?uorescence reporters with advanced properties is one of the most important problems. In this volume, top experts in this ?eld provide advanced knowledge on the design and properties of ?uorescent dyes. Organic dyes were the ?rst ?uorescent materials used for analytical purposes, and we observe that they retain their leading positions against strong competition of new materials - conjugated polymers, semiconductor nanocrystals, and metal chelating complexes. Recently, molecular and cellular biology got a valuable tool of organic ?uorophores synt- sized by cell machinery and incorporated into green ?uorescent protein and its analogs. Demands of various ?uorescence techniques operating in spectral, anisotropy, and time domains require focused design of ?uorescence reporters well adapted to these techniques. Near-IR spectral range becomes more and more attractive for various applications, and new dyes emitting in this range are strongly requested. Two-photonic ?uorescence has become one of the major tools in bioimaging, and ?uorescence reporters well adapted to this technique are in urgent need. These problems cannot be solved without the knowledge of fundamental principles of dye design and of physical phenomena behind their ?uorescence response.

This book provides systematic knowledge of basic principles in the design of fluorescence sensing and imaging techniques together with critical analysis of recent developments. Fluorescence is the most popular technique in chemical and biological sensing because of its ultimate sensitivity, high temporal and spatial resolution and versatility that enables imaging within the living cells. It develops rapidly in the directions of constructing new molecular recognition units, new fluorescence reporters and in improving sensitivity of response up to detection of single molecules. Its application areas range from control of industrial processes to environment monitoring and clinical diagnostics. Being a guide for students and young researchers, it also addresses professionals involved in active basic and applied research. Making a strong link between education, research and product development, this book discusses prospects for future progress.

This book provides systematic knowledge of basic principles in the design of fluorescence sensing and imaging techniques together with critical analysis of recent developments. Fluorescence is the most popular technique in chemical and biological sensing because of its ultimate sensitivity, high temporal and spatial resolution and versatility that enables imaging within the living cells. It develops rapidly in the directions of constructing new molecular recognition units, new fluorescence reporters and in improving sensitivity of response up to detection of single molecules. Its application areas range from control of industrial processes to environment monitoring and clinical diagnostics. Being a guide for students and young researchers, it also addresses professionals involved in active basic and applied research. Making a strong link between education, research and product development, this book discusses prospects for future progress.

The key element of any fluorescence sensing or imaging technology is the fluorescence reporter, which transforms the information on molecular interactions and dynamics into measurable signals of fluorescence emission. This book, written by a team of frontline researchers, demonstrates the broad field of applications of fluorescence reporters, starting from nanoscopic properties of materials, such as self-assembled thin films, polymers and ionic liquids, through biological macromolecules and further to living cell, tissue and body imaging. Basic information on obtaining and interpreting experimental data is presented and recent progress in these practically important areas is highlighted. The book is addressed to a broad interdisciplinary audience.

Avarietyof?uorescentandluminescentmaterialsintheformofmolecules,their complexes,andnanoparticlesareavailableforimplementationasreportingunits intosensingtechnologies. Increasingdemandsfromtheseapplicationareasrequire developmentofnew?uorescencereportersbasedonassociationandaggregationof ?uorescencedyesandontheirincorporationintodifferentnanostructures. Inter- tionsbetweenthesedyesandtheirincorporatingmatricesleadtonewspectroscopic effectsthatcanbeactivelyusedforoptimizingthesensordesign. Oneofthese effects is a spectacular formation of J-aggregates with distinct and very sharp excitationandemissionbands. Byincorporationintonanoparticles,organicdyes offer dramatically increased brightness together with improvement of chemical stabilityandphotostability. Moreover,certaindyescanformnanoparticlesth- selvessothattheirspectroscopicpropertiesareimproved. Semiconductorquantum dotsaretheothertypeofnanoparticles thatpossessuniqueandveryattractive photophysicalandspectroscopicproperties. Manyinterestingandnotfullyund- stoodphenomenaareobservedinclusterscomposedofonlyseveralatomsofnoble metals. Inconjugatedpolymers,strongelectronicconjugationbetweenelementary chromophoricunitsresultsindramaticeffectsinquenchingandinconformati- dependentspectroscopicbehavior. Possessingsuchpowerfulanddiversearsenaloftools,wehavetoexplorethem innovelsensingandimagingtechnologiesthatcombineincreasedbrightnessand sensitivityinanalytedetectionwithsimplicityandlowcostofproduction. The present book overviews the pathways for achieving this goal. In line with the discussion on monomeric ?uorescence reporters in the accompanying book (Vol. 8ofthisseries),aninsightfulanalysisofphotophysicalmechanismsbehind the ?uorescence response of composed and nanostructured materials is made. Based on the progress in understanding these mechanisms, their realization in differentchemicalstructuresisoverviewed. vii viii Preface Demonstratingtheprogressinaninterdisciplinary?eldofresearchanddev- opment,thisbookisprimarilyaddressedtospecialistswithdifferentbackground- physicists, organic and analytical chemists, and photochemists - to those who developandapplynew?uorescencereporters. Itwillalsobeusefultospecialists inbioanalysisandbiomedicaldiagnostics. Kyiv,Ukraine AlexanderP. Demchenko June2010 Contents PartI GeneralAspects NanocrystalsandNanoparticlesVersusMolecularFluorescent LabelsasReportersforBioanalysisandtheLifeSciences: ACriticalComparison ...3 UteResch-Genger,MarkusGrabolle,RolandNitschke, andThomasNann OptimizationoftheCouplingofTargetRecognition andSignalGeneration ...41 AnaB. Descalzo,ShengchaoZhu,TobiasFischer,andKnutRurack CollectiveEffectsIn?uencingFluorescenceEmission ...107 AlexanderP. Demchenko PartII EncapsulatedDyesandSupramolecularConstructions FluorescentJ-AggregatesandTheirBiologicalApplications ...135 MykhayloYu. LosytskyyandValeriyM. Yashchuk Conjugates,Complexes,andInterlockedSystems BasedonSquarainesandCyanines ...159 LeonidD. Patsenker,AnatoliyL. Tatarets,OleksiiP. Klochko, andEwaldA. Terpetschnig PartIII Dye-DopedNanoparticlesandDendrimers Dye-DopedPolymericParticlesforSensingandImaging ...193 SergeyM. Borisov,TorstenMayr,Gu..nterMistlberger,andIngoKlimant ix x Contents Silica-BasedNanoparticles:DesignandProperties ...229 SongLiang,CarrieL. John,ShupingXu,JiaoChen,YuhuiJin, QuanYuan,WeihongTan,andJuliaX. Zhao LuminescentDendrimersasLigandsandSensors ofMetalIons ...2 53 GiacomoBergamini,EnricoMarchi,andPaolaCeroni ProspectsforOrganicDyeNanoparticles ...285 HiroshiYao PartIV LuminescentMetalNanoclusters Few-AtomSilverClustersasFluorescentReporters ...307 IsabelD?'ezandRobinH. A. Ras LuminescentQuantumClustersofGoldasBio-Labels ...333 M. A. HabeebMuhammedandT. Pradeep PartV ConjugatedPolymers Structure,EmissiveProperties,andReportingAbilities ofConjugatedPolymers ...357 MaryA. Reppy OpticalReportingbyConjugatedPolymers viaConformationalChanges ...389 RozalynA. SimonandK. PeterR. Nilsson FluorescenceReportingBasedonFRETBetweenConjugated PolyelectrolyteandOrganicDyeforBiosensorApplications ...417 Kan-YiPuandBinLiu Index ...455 PartI GeneralAspects NanocrystalsandNanoparticlesVersus MolecularFluorescentLabelsasReporters forBioanalysisandtheLifeSciences: ACriticalComparison UteResch-Genger,MarkusGrabolle,RolandNitschke,andThomasNann Abstract At the core of photoluminescence techniques are suitable ?uorescent labels and reporters, the spectroscopic properties of which control the limit of detection,thedynamicrange,andthepotentialformultiplexing.

Fluorescence reporter is the key element of any sensing or imaging technology. Its optimal choice and implementation is very important for increasing the sensitivity, precision, multiplexing power, and also the spectral, temporal, and spatial reso- tion in different methods of research and practical analysis. Therefore, design of ?uorescence reporters with advanced properties is one of the most important problems. In this volume, top experts in this ?eld provide advanced knowledge on the design and properties of ?uorescent dyes. Organic dyes were the ?rst ?uorescent materials used for analytical purposes, and we observe that they retain their leading positions against strong competition of new materials - conjugated polymers, semiconductor nanocrystals, and metal chelating complexes. Recently, molecular and cellular biology got a valuable tool of organic ?uorophores synt- sized by cell machinery and incorporated into green ?uorescent protein and its analogs. Demands of various ?uorescence techniques operating in spectral, anisotropy, and time domains require focused design of ?uorescence reporters well adapted to these techniques. Near-IR spectral range becomes more and more attractive for various applications, and new dyes emitting in this range are strongly requested. Two-photonic ?uorescence has become one of the major tools in bioimaging, and ?uorescence reporters well adapted to this technique are in urgent need. These problems cannot be solved without the knowledge of fundamental principles of dye design and of physical phenomena behind their ?uorescence response.

The aim of this book is to give a comprehensive description of the basic methods used in the ultraviolet spectroscopy of proteins, to discuss new trends and development of these methods, and to analyze their different applications in the study of various aspects of protein structure and dynamics. Ultraviolet spectroscopy is one of the oldest and most popular methods in the field of biochemistry and molecular biophysics. At present, it is difficult to imagine the biochemical laboratory without a recording spectrophotometer or spectrofluorimeter. There are several hundreds of publications directly devoted to protein ultraviolet spectroscopy and in a great number of studies UV spectroscopic methods are used for the structural analysis of different proteins. Meanwhile a unified description of the theoretical basis of the methods, experimental techniques, data analysis, and generalization of results obtained in solving the specific problems of protein structure are lacking. There are three reasons for which a monograph on ultraviolet spectroscopy is needed today. Firstly, there has been significant growth in facilities of experimental technique, its precision, and versatility associated with computer data analysts. This new technique is available to a wide circle of scientists engaged in the field of protein research. Most of them are not spectroscopists and, thus, there is a need for a conceivable and precise source of information on how to use this method and what kind of data it should provide.

The key element of any fluorescence sensing or imaging technology is the fluorescence reporter, which transforms the information on molecular interactions and dynamics into measurable signals of fluorescence emission. This book, written by a team of frontline researchers, demonstrates the broad field of applications of fluorescence reporters, starting from nanoscopic properties of materials, such as self-assembled thin films, polymers and ionic liquids, through biological macromolecules and further to living cell, tissue and body imaging. Basic information on obtaining and interpreting experimental data is presented and recent progress in these practically important areas is highlighted. The book is addressed to a broad interdisciplinary audience.

Avarietyof?uorescentandluminescentmaterialsintheformofmolecules,their complexes,andnanoparticlesareavailableforimplementationasreportingunits intosensingtechnologies. Increasingdemandsfromtheseapplicationareasrequire developmentofnew?uorescencereportersbasedonassociationandaggregationof ?uorescencedyesandontheirincorporationintodifferentnanostructures. Inter- tionsbetweenthesedyesandtheirincorporatingmatricesleadtonewspectroscopic effectsthatcanbeactivelyusedforoptimizingthesensordesign. Oneofthese effects is a spectacular formation of J-aggregates with distinct and very sharp excitationandemissionbands. Byincorporationintonanoparticles,organicdyes offer dramatically increased brightness together with improvement of chemical stabilityandphotostability. Moreover,certaindyescanformnanoparticlesth- selvessothattheirspectroscopicpropertiesareimproved. Semiconductorquantum dotsaretheothertypeofnanoparticles thatpossessuniqueandveryattractive photophysicalandspectroscopicproperties. Manyinterestingandnotfullyund- stoodphenomenaareobservedinclusterscomposedofonlyseveralatomsofnoble metals. Inconjugatedpolymers,strongelectronicconjugationbetweenelementary chromophoricunitsresultsindramaticeffectsinquenchingandinconformati- dependentspectroscopicbehavior. Possessingsuchpowerfulanddiversearsenaloftools,wehavetoexplorethem innovelsensingandimagingtechnologiesthatcombineincreasedbrightnessand sensitivityinanalytedetectionwithsimplicityandlowcostofproduction. The present book overviews the pathways for achieving this goal. In line with the discussion on monomeric ?uorescence reporters in the accompanying book (Vol. 8ofthisseries),aninsightfulanalysisofphotophysicalmechanismsbehind the ?uorescence response of composed and nanostructured materials is made. Based on the progress in understanding these mechanisms, their realization in differentchemicalstructuresisoverviewed. vii viii Preface Demonstratingtheprogressinaninterdisciplinary?eldofresearchanddev- opment,thisbookisprimarilyaddressedtospecialistswithdifferentbackground- physicists, organic and analytical chemists, and photochemists - to those who developandapplynew?uorescencereporters. Itwillalsobeusefultospecialists inbioanalysisandbiomedicaldiagnostics. Kyiv,Ukraine AlexanderP. Demchenko June2010 Contents PartI GeneralAspects NanocrystalsandNanoparticlesVersusMolecularFluorescent LabelsasReportersforBioanalysisandtheLifeSciences: ACriticalComparison ...3 UteResch-Genger,MarkusGrabolle,RolandNitschke, andThomasNann OptimizationoftheCouplingofTargetRecognition andSignalGeneration ...41 AnaB. Descalzo,ShengchaoZhu,TobiasFischer,andKnutRurack CollectiveEffectsIn?uencingFluorescenceEmission ...107 AlexanderP. Demchenko PartII EncapsulatedDyesandSupramolecularConstructions FluorescentJ-AggregatesandTheirBiologicalApplications ...135 MykhayloYu. LosytskyyandValeriyM. Yashchuk Conjugates,Complexes,andInterlockedSystems BasedonSquarainesandCyanines ...159 LeonidD. Patsenker,AnatoliyL. Tatarets,OleksiiP. Klochko, andEwaldA. Terpetschnig PartIII Dye-DopedNanoparticlesandDendrimers Dye-DopedPolymericParticlesforSensingandImaging ...193 SergeyM. Borisov,TorstenMayr,Gu..nterMistlberger,andIngoKlimant ix x Contents Silica-BasedNanoparticles:DesignandProperties ...229 SongLiang,CarrieL. John,ShupingXu,JiaoChen,YuhuiJin, QuanYuan,WeihongTan,andJuliaX. Zhao LuminescentDendrimersasLigandsandSensors ofMetalIons ...2 53 GiacomoBergamini,EnricoMarchi,andPaolaCeroni ProspectsforOrganicDyeNanoparticles ...285 HiroshiYao PartIV LuminescentMetalNanoclusters Few-AtomSilverClustersasFluorescentReporters ...307 IsabelD?'ezandRobinH. A. Ras LuminescentQuantumClustersofGoldasBio-Labels ...333 M. A. HabeebMuhammedandT. Pradeep PartV ConjugatedPolymers Structure,EmissiveProperties,andReportingAbilities ofConjugatedPolymers ...357 MaryA. Reppy OpticalReportingbyConjugatedPolymers viaConformationalChanges ...389 RozalynA. SimonandK. PeterR. Nilsson FluorescenceReportingBasedonFRETBetweenConjugated PolyelectrolyteandOrganicDyeforBiosensorApplications ...417 Kan-YiPuandBinLiu Index ...455 PartI GeneralAspects NanocrystalsandNanoparticlesVersus MolecularFluorescentLabelsasReporters forBioanalysisandtheLifeSciences: ACriticalComparison UteResch-Genger,MarkusGrabolle,RolandNitschke,andThomasNann Abstract At the core of photoluminescence techniques are suitable ?uorescent labels and reporters, the spectroscopic properties of which control the limit of detection,thedynamicrange,andthepotentialformultiplexing.