Springer Series on Fluorescence 15 Series Editor: Martin Hof Peter Kapusta Michael Wahl Rainer Erdmann Editors Advanced Photon Counting Applications, Methods, Instrumentation 15 Springer Series on Fluorescence Methods and Applications Series Editor: Martin Hof Springer Series on Fluorescence Series Editor: Martin Hof Recently Published and Forthcoming Volumes AdvancedPhotonCounting LanthanideLuminescence VolumeEditors:PeterKapusta, Photophysical,AnalyticalandBiologicalAspects MichaelWahlandRainerErdmann VolumeEditors:P.Ha¨nninenandH.Ha¨rma¨ Vol.15,2015 Vol.7,2011 Far-FieldOpticalNanoscopy StandardizationandQualityAssurance VolumeEditors:PhilipTinnefeld,Christian inFluorescenceMeasurementsII EggelingandStefanW.Hell BioanalyticalandBiomedicalApplications Vol.14,2015 VolumeEditor:Resch-Genger,U. 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Wahl VolumeEditors PeterKapusta MichaelWahl J.HeyrovskyInstituteofPhysical PicoQuantGmbH Chemistry Berlin AcademyofSciencesoftheCzech Germany Republic Prague8 CzechRepublic RainerErdmann PicoQuantGmbH Berlin Germany ISSN1617-1306 ISSN1865-1313 (electronic) SpringerSeriesonFluorescence ISBN978-3-319-15635-4 ISBN978-3-319-15636-1 (eBook) DOI10.1007/978-3-319-15636-1 LibraryofCongressControlNumber:2015938183 SpringerChamHeidelbergNewYorkDordrechtLondon #SpringerInternationalPublishingSwitzerland2015 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionor informationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar methodologynowknownorhereafterdeveloped. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publicationdoesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexempt fromtherelevantprotectivelawsandregulationsandthereforefreeforgeneraluse. Thepublisher,theauthorsandtheeditorsaresafetoassumethattheadviceandinformationinthisbook arebelievedtobetrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsorthe editorsgiveawarranty,expressorimplied,withrespecttothematerialcontainedhereinorforanyerrors oromissionsthatmayhavebeenmade. Printedonacid-freepaper Springer International Publishing AG Switzerland is part of Springer Science+Business Media (www.springer.com) Series Editor Prof.Dr.MartinHof AcademyofSciencesoftheCzechRepublic J.HeyrovskyInstituteofPhysicalChemistry DepartmentofBiophysicalChemistry Dolejskova3 16223Prague8 CzechRepublic [email protected] Aims and Scope Fluorescencespectroscopy,fluorescenceimagingandfluorescentprobesareindis- pensible tools in numerous fields of modern medicine and science, including molecularbiology,biophysics,biochemistry,clinicaldiagnosisandanalyticaland environmentalchemistry.Applicationsstretchfromspectroscopyandsensortech- nology to microscopy and imaging, to single molecule detection, to the develop- ment of novel fluorescent probes, and to proteomics and genomics. The Springer SeriesonFluorescenceaimsatpublishingstate-of-the-artarticlesthatcanserveas invaluable tools for both practitioners and researchers being active in this highly interdisciplinaryfield.Thecarefullyeditedcollectionofpapersineachvolumewill givecontinuousinspirationfornewresearchandwillpointtoexcitingnewtrends. . Preface Getherexcitedredorgreen; letitdependonwhatshe’skeen. Quitesoonquiteradiantshewillbeseen; incolourstrueofhershe’llgleam, butmindthenanosecondsinbetween. Fortheymighttellwhichwaytolean: perhapsshe’sotherthanyouhadforeseen. Although,bepatientwhenyoutrythisscheme; asingleflashthatlightsthescene isnotenoughtogethertheme. Infact,hermoodsaresoextreme thattrulyrandomshemightseem. Andyet,ifpatientlyyousiftherstream apatternoftruebeautycanbeseen. Sosumyourcountandbeserene. In1926thephysicistFrithiofWolfersandthechemistGilbertN.Lewiscoinedthe namephotonforthequantumoflightdiscoveredabout20yearsearlier.Evenifit maylookalittlesuperficialatfirstglance,letusnotetheinvolvementofchemists hereandelsewhereintheevolutionofquantumphysics.Indeed,theoverwhelming successofquantummechanicsasamodernscientifictheoryisrootednotsomuch inpurephysicsbutinitsinescapablyconvincingexplanatorypowerforvirtuallyall aspectsofphysicalchemistryandmaterialscience.Modellingatomsandmolecules as quantum mechanical systems undergoing transitions between quantum states, some of them involving photon absorption and emission, was the key to under- standing and eventually even exploiting virtually all previously mysterious spec- troscopiceffects.Inthissenseitisnotasurprisethatthemethodsaddressedinthis volume are now used more often in chemistry and related fields than in pure physics.Infact,spectroscopicmethodshavebecomeindispensibleinbiochemistry because light as a probe, suitably applied, can be used in living cells without any damage to the specimen and without unduly spoiling the functions or processes vii viii Preface under investigation. Even though classic spectroscopy does not require working withsinglephotons,itturnsoutveryusefulthatitcanbedoneso.Thisisthecase when there are only very few molecules involved in the processes of interest, in particularinthevitalprocessesexploredbymolecularbiology.Evenmoreinterest- ingthanclassicspectroscopyisthescenariowhereyetanotherquantummechanical propertyisused:thelifetimeoftheexcitedstate.Itturnsoutthattheaveragetimea moleculespendsinthisstateissospecifictothatmoleculeanditsenvironmentthat itcanbeusedasafingerprintofthemoleculeinadditiontoitsspectrumand/orasa probeforcertainenvironmentparameters.Eventhoughthelifetimeofanindividual excited state is completely unpredictable according to quantum mechanics, the averagelifetimeisbothmeasurableandmeaningful.Inpracticeitcanbeobserved asthedurationofluminescencefromanensembleofmoleculesexcitedbyashort flash of light. Going by the observed phenomenon one speaks of fluorescence or phosphorescencelifetimemeasurements.Inthatcasethenecessaryaveragingofthe individualexcitedstatelifetimesisachievedimplicitlybecauseofthesimultaneous observationofphotonsfromtheentireensemble.Theotherinterestingscenariois thatofaveragingtheexcitedstatelifetimeacrossmultiplecyclesofexcitationand photonemissionobservedonjustonemolecule.Indeed,byvirtueofergodicity,this kindofmeasurementgivesthesameresultsastheensemblemeasurement.Inthis case time-correlated single photon counting (TCSPC) is the method of choice. It allowsluminescencelifetimemeasurementsonsinglemoleculesandotherisolated quantumsystems.Combining spectralinformationand,e.g.,fluorescencelifetime onecanusethisrefined“fingerprint”ofthemoleculesofinteresttoidentifythem even inthepresence ofsignificant background. Itwaslargelythisidea,combined withconfocaldetection,whichfinallyledtotheincredibleachievementsinsingle moleculedetection,singlemoleculespectroscopy,andevenmicroscopybymeans of TCSPC. Powerful methods such as the exploitation of Fo¨rster resonant energy transfer (FRET) asa molecularruler became routine toolsfor the investigationof protein folding and interaction when they were made accessible for single mole- culesalongthisway. However,TCSPCisnotonlyusefulwithsinglemolecules.Aswewillshowin thefirstchaptersofthepresentvolume,italsohelpstoachievebettertimeresolu- tion with typical detectors, even in ensemble measurements. These technology related chapters will cover the state of the art of current hardware, hopefully also convincing the reader that despite the inherent statistical nature of the measure- ment, modern users can rely on incredibly fast instruments and must not be as patientasourlittleold-fashionedopeningpoemmightsuggest.Indeeditismostly thememoryof“oldfashioned”instrumentationthatlefttheimpressionofslowness attached to TCSPC. This may be illustrated by personal memories held probably not only by the editors: “I recall with nostalgia the long nights spent alone in a dimmedbasementlabwaitingforcollectionofatleast1000countsinthepeakby our beautiful TCSPC monster powered by a 45 kHz flashlamp.” This is the past. Meanwhile things have changed dramatically, not only because of faster TCSPC electronicsbutverymuchalsobecauseoffaster(andeasiertouse)lasers,towhich achapterinitsownwillbededicated.Theremainderofthechaptersarecoveringa Preface ix rich eclectic mixture of application topics as well as methodology in experiment anddataanalysis.Despiteoftheimportanceoflifescienceapplicationswetriedto embrace a much wider scope, including, e.g., defect centers in diamond as single photon sources and quantum sensors, as well as optical tomography and super resolutionmicroscopy.Similarly,onthemethodologyandinstrumentationside,we aimedtoshowtheinterestingnewoptionsarisingfromthecombinationofappar- ently distinct methods such as classic TCSPC and fluorescence lifetime with methods based on intensity fluctuation. Together with the authors, to whom we expressourgratitudehere,wehopetoprovideavolumeofbothanimmediatevalue as a current overview of the field and some longer term value as a collection of referencetexts. Prague,CzechRepublic PeterKapusta Berlin,Germany MichaelWahl RainerErdmann