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Springer Series on Fluorescence 14 Series Editor: Otto S. Wolfbeis Philip Tinnefeld Christian Eggeling Stefan W. Hell Editors Nobel Prize 2014 Far-Field Optical Nanoscopy 14 Springer Series on Fluorescence Methods and Applications Series Editor: Otto S. Wolfbeis Springer Series on Fluorescence Series Editor: Otto S. Wolfbeis Recently Published and Forthcoming Volumes Far-FieldOpticalNanoscopy LanthanideLuminescence VolumeEditors:PhilipTinnefeld,Christian Photophysical,AnalyticalandBiologicalAspects EggelingandStefanW.Hell VolumeEditors:P.Ha¨nninenandH.Ha¨rma¨ Vol.14,2015 Vol.7,2011 FluorescentMethodstoStudyBiological StandardizationandQualityAssurance Membranes inFluorescenceMeasurementsII VolumeEditors:Y.Me´lyandG.Duportail BioanalyticalandBiomedicalApplications Vol.13,2013 VolumeEditor:Resch-Genger,U. Vol.6,2008 FluorescentProteinsII ApplicationofFluorescentProteinTechnology StandardizationandQualityAssurance VolumeEditor:G.Jung inFluorescenceMeasurementsI Vol.12,2012 Techniques VolumeEditor:U.Resch-Genger FluorescentProteinsI Vol.5,2008 FromUnderstandingtoDesign VolumeEditor:G.Jung FluorescenceofSupermolecules,Polymeres, Vol.11,2012 andNanosystems VolumeEditor:M.N.Berberan-Santos AdvancedFluorescenceReporters Vol.4,2007 inChemistryandBiologyIII ApplicationsinSensingandImaging FluorescenceSpectroscopyinBiology VolumeEditor:A.P.Demchenko VolumeEditor:M.Hof Vol.10,2011 Vol.3,2004 AdvancedFluorescenceReportersin FluorescenceSpectroscopy,Imaging ChemistryandBiologyII andProbes MolecularConstructions,Polymersand VolumeEditor:R.Kraayenhof Nanoparticles Vol.2,2002 VolumeEditor:A.P.Demchenko NewTrendsinFluorescenceSpectroscopy Vol.9,2010 VolumeEditor:B.Valeur Vol.1,2001 AdvancedFluorescenceReporters inChemistryandBiologyI FundamentalsandMolecularDesign VolumeEditor:A.P.Demchenko Vol.8,2010 Moreinformationaboutthisseriesat http://www.springer.com/series/4243 Far-Field Optical Nanoscopy Volume Editors: Philip Tinnefeld Christian Eggeling Stefan W. Hell With contributions by G.P.Acuna(cid:1)P.F.Aramend´ıa(cid:1)M.L.Bossi(cid:1)M.Brameshuber(cid:1) (cid:1) (cid:1) (cid:1) (cid:1) M.P. Bruchez T. Cordes G. De Cremer D.E. De Vos (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) C.Eggeling C.Forthmann S.Gayda A.Gietl P.N.Hedde (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) S.W. Hell J. Hofkens B. Lalkens S. Laurien S.F. Lee (cid:1) (cid:1) (cid:1) (cid:1) H.-l.D. Lee M.D. Lew W.E. Moerner K. Nienhaus (cid:1) (cid:1) (cid:1) (cid:1) G.U.Nienhaus M.B.J.Roeffaers M.Sauer J.J.Schmied G.J. Schu¨tz (cid:1) B.F. Sels (cid:1) S.-H. Shim (cid:1) I.H. Stein (cid:1) (cid:1) (cid:1) (cid:1) C. Steinhauer M.A. Thompson P. Tinnefeld (cid:1) (cid:1) (cid:1) (cid:1) M.H. Ulbrich J. Vogelsang K. Xu D.A. Yushchenko X. Zhuang VolumeEditors PhilipTinnefeld ChristianEggeling InstituteofPhysicalandTheoretical MRCHumanImmunologyUnitand Chemistry–NanoBioSciences WolfsonImagingCentreOxford TUBraunschweig WeatherallInstituteofMolecular Braunschweig Medicine Germany UniversityofOxford Oxford StefanW.Hell UnitedKingdom DepartmentofNanoBiophotonics MaxPlanckInstituteforBiophysical Chemistry Go¨ttingen Germany DivisionofOpticalNanoscopy GermanCancerResearchCenter Heidelberg Germany ISSN1617-1306 ISSN1865-1313(electronic) ISBN978-3-662-45546-3 ISBN978-3-662-45547-0(eBook) DOI10.1007/978-3-662-45547-0 SpringerHeidelbergNewYorkDordrechtLondon LibraryofCongressControlNumber:2015930089 #Springer-VerlagBerlinHeidelberg2015 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartof the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,broadcasting,reproductiononmicrofilmsorinanyotherphysicalway,andtransmissionor informationstorageandretrieval,electronicadaptation,computersoftware,orbysimilarordissimilar methodologynowknownorhereafterdeveloped.Exemptedfromthislegalreservationarebriefexcerpts inconnectionwithreviewsorscholarlyanalysisormaterialsuppliedspecificallyforthepurposeofbeing enteredandexecutedonacomputersystem,forexclusiveusebythepurchaserofthework.Duplication ofthispublicationorpartsthereofispermittedonlyundertheprovisionsoftheCopyrightLawofthe Publisher’s location, in its current version, and permission for use must always be obtained from Springer.PermissionsforusemaybeobtainedthroughRightsLinkattheCopyrightClearanceCenter. ViolationsareliabletoprosecutionundertherespectiveCopyrightLaw. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublication doesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevant protectivelawsandregulationsandthereforefreeforgeneraluse. Whiletheadviceandinformationinthisbookarebelievedtobetrueandaccurateatthedateofpublication, neithertheauthorsnortheeditorsnorthepublishercanacceptanylegalresponsibilityforanyerrorsor omissionsthatmaybemade.Thepublishermakesnowarranty,expressorimplied,withrespecttothe materialcontainedherein. Printedonacid-freepaper SpringerispartofSpringerScience+BusinessMedia(www.springer.com) Series Editor Prof.Dr.OttoS.Wolfbeis InstituteofAnalyticalChemistry Chemo-andBiosensors UniversityofRegensburg 93040Regensburg Germany [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 development 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 Live-cell investigations to a great extent rely on far-field optical microscopy techniquessuchaswide-field,orconfocalmicroscopy,becausefluorescencedetec- tionoftaggedmoleculessuchasproteinsofferslargespecificityandsensitivityand lens-based far-field optics allows the observation of the cellular interior with minimal invasion. Over the centuries, major efforts have been exerted toimprove theresolutionoffar-fieldlightmicroscopy,whichforlongmeantperfectinglenses and other optical elements. The limits of this strategy became evident with the discoveryofthediffractionbarrierinthenineteenthcentury.Fromthenonwards,it was clear that light cannot be focused to a spot smaller than about half the wavelength of light, i.e., to about 200–300 nm. This meant that molecules, such asproteins,oranyotherfeaturesthatareclosertogetherthanthisrange,cannotbe discerned.Forexample,proteindistributionscannotbevisualizedatsmallerscales inacell.Thediffractionbarrierclearlyimpededtheapplicabilityoffar-fieldoptical microscopy in the life sciences. Therefore, over the decades, efforts have been undertakentoovercomethismajorlimitation,butitwasnotuntilattheendofthe twentiethcenturythatthisbarrierwasovercome.Sincethen,amultitudeofsuper- resolution far-field fluorescence microscopy or nanoscopy techniques have been developed,suchasSTED,GSD,RESOLFT,(f)PALM,(d)STORM,GSDIM,etc., and it has become clear that lens-based fluorescence microscopes can resolve featuresatthenanometerscale. Attheirverybasis,allcurrentfluorescencenanoscopytechniquessharethesame operationalprinciple:nearbysamplemoleculesarenolongerdiscernedjustbythe phenomenon of focusing light but by prompting them to briefly assume (at least) twodifferentstates.Usually, the(two) statesthatare selected forseparationarea fluorescence on- and an off-state. In other words, turning molecules on and off is employedsothatthesignaldoesnotstemfromallmoleculessimultaneously.Thus, the use of two different states for molecular distinction renders nearby objects distinguishable when illuminated by the same diffraction pattern, yieldingimages withnanoscaleresolution. Unfortunately, even up to date, there is a lot of confusion about the basics, similarities,anddifferencesofthevariousnanoscopyapproaches,aswellasabout vii viii Preface their potential. Parts of these concerns are based on contingent issues such as an increased setup complexity, phototoxicity, and demands on labeling. Yet, recent years have seen major improvements, allowing the widespread onset of turn-key opticalnanoscopesintoopenfacilitiesofbiologicalinstitutes.Here,amajordrive has been the developmentsinlasers andoptics technology butspecifically alsoin labelingtechniques. Asamatteroffact,inopticalnanoscopy,fluorescentlabelsmustfulfilltworoles. First,theyhavetohighlightthestructuresofinterest,givingbrightsignalthatmay bedistinguishablefromothermoleculartags.Secondly,thelabelshavetoprovide thepairofdifferentstates,saytheon-andoff-statesrequiredfordiscerningnearby molecules.Therefore,itisthesymbiosisofmoleculartagsandopticaldesignthat makesthemicroscopesoftodaysharpandbright. Thisbooktacklesalloftheaboveissues.In11chaptersdistinguishedscientists and leaders in the respective fields elaborate on the basics of the different nano- scopyapproachesandtheirrecentadvancements,onimprovedlabelingtechnology, aswellasgiveanoverviewofrepresentativeapplications. The first part of the book introduces different optical nanoscopy approaches. Chapter“STEDFluorescence Nanoscopy”(byC.EggelingandS.W.Hell)introduces STED microscopy, the earliest far-field fluorescence nanoscopy approach. Thechapterdescribestheadvancesthathaveovercomeinitialsystemcomplexities, resulting in multiple applications of this technique. Chapter “Super-Resolution ImagingThrough Stochastic Switching and Localization of Single Molecules: An Overview”byX.Zhuangandco-workersintroducestheSTORMapproach,guiding thereaderthroughthebasicsandselectedapplicationsofthistechniqueandofthe related approaches (f)PALM, GSDIM, etc. In chapter “A Practical Guide to dSTORM: Super-Resolution Imaging with Standard Fluorescent Probes”, the approach called dSTORM by M. Sauer is presented. Similar to GSDIM or blink microscopy,thegreatachievementherewastheinvestigationofhowtoimplement anyorganicdyeorfluorescentproteinintotheconceptofSTORM/(f)PALM,which were initiallyrestrictedtoacertain class ofphotoswitchable dyes.Specifically,in dSTORM dedicated buffer conditions for mounting of samples have been developed. Thesecondpartofthebookfocusesonthedemandsthatopticalnanoscopyhas putonthe fluorescence labels.Chapter“Single-Molecule PhotocontrolandNano- scopy” by W. E. Moerner and co-workers depicts the development of eYFP and novel photoactivatable organic dyes for their use in (d)STORM/(f)PALM type of nanoscopy.Chapter“ProbesforNanoscopy:FluorescentProteins”byU.Nienhaus andco-workersprovides anoverview offluorescent proteins,with aspecial focus on their usage in optical nanoscopy. In chapter “Tailoring Fluorescent Labels for Far-Field Nanoscopy”, D. A. Yushchenko and M. P. Bruchez highlight the advancesinfluorophoredevelopmentforarangeofexistingtechniques.Emphasis isplacedontherequirementsthesedistinctmethodsdemandfromthefluorophores inordertomaximizeresolution,particularlyinlive-cellimaging.P.F.Aramendia andM.L.Bossi(chapter“ProbesforNanoscopy:PhotoswitchableFluorophores”) discuss on how photochromic organic dyes can be designed to fulfill the Preface ix requirementsofasuitableprobefordifferentfluorescencenanoscopystrategies.In chapter “Far-Field Nanoscopy with Conventional Fluorophores: Photostability, Photophysics,andTransientBinding”,P.Tinnefeldandhisteamgiveanoverview oftheirworkonusingconventionalorganicfluorophoresfor(d)STORM/(f)PALM type nanoscopy, with a special focus on the importance and principles of photo- stabilityandblinkingcharacteristicsoftheseprobes. Thethirdpartgivesspecificdevelopmentsandselectedapplications.Inchapter “NASCAMicroscopy:Super-ResolutionMappingofChemicalReactionCenters” J. Hofkens, B.J. Roeffaers and co-workers introduce their concept of NASCA nanoscopy to visualize single chemical reaction centers with sub-diffraction reso- lutionusingspecialfluorogenicprobemolecules.M.H.Ulbrich(chapter“Counting Molecules: Toward Quantitative Imaging”) shows how single-molecule fluorescence can be used to analyze protein–protein interactions by enabling the direct visualizationofproteincomplexesandthenumberandspeciesoftheirconstituent subunits. Finally, in chapter “In Vivo Tracking of Single Biomolecules: What Trajectories Tell Us About the Acting Forces” M. Brameshuber and G. J. Schu¨tz introducethereadertotheconceptofthetrackingofsinglebiomoleculesinliving cells,andhowtheirtrajectoriescanreportonactingforces,e.g.,bytheunderlying cytoskeleton. Emerging nanoscopy techniques are flourishing and have started revealing exciting new biology, such as insights of the cytoskeleton, protein complexes, or virusmaturation.Meanwhile,fluorescencenanoscopesarecommerciallyavailable andnewprobesaredevelopedatfastpace.Thecollectionofchaptersinthisbook can present only a glimpse on these exciting developments. The work presented, however,givesagoodoverviewintodifferentapproachesofthisinterdisciplinary andrapidlydevelopingfield.Fluorescencenanoscopyinvolvesanewphysicsand new chemistry to reveal new biology! We are grateful to the authors for their outstanding contributions and believe that the book will be stimulating for the communityofmicroscopedeveloper,microscopyusers,andallinterestedreaders. Braunschweig,Germany PhilipTinnefeld Oxford,UK ChristianEggeling Go¨ttingen,Germany StefanW.Hell