Table Of ContentSpringer 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
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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
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)intro-
duces 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 ap-
proach 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 devel-
oped.
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 fluores-
cence 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
