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Maximizing far-field optical microscopy resolution through selected fluorophore transitions [Elektronische Ressource] / put forward by Eva Rittweger

94 pages
Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural SciencesPut forward byDiplom-Physikerin Eva Rittwegerborn in ColognethOral examination: 16 December 2009Maximizing far-field opticalmicroscopy resolution throughselected fluorophore transitions0Referees: Prof. Dr. Stefan W. HellProf. Dr. Ju¨rgen WolfrumAbstract:Stimulated emission depletion (STED) microscopy and related nanoscopy tech-niques, which utilize a saturable optical transition between a bright and a darkstate, overcome the diffraction barrier by confining one of the states to an areasmaller than the Airy disk. Scanning this area across the specimen yields sub-diffraction images by registering inseparable fluorescent markers sequentially intime. Despite the progress made in nanoscopy so far, maximizing the resolu-tion has been hampered by the efficiency of the utilized optical transition andthe photostability of the fluorophores. Here, the optical transition responsiblefor breaking the barrier was studied in order to maximize its efficiency. For arange of fluorophores (dyes, proteins, quantum dots, color centers) the nature ofthe responsible process could be clarified. It was also investigated whether heatcould serve as an imaging contrast to provide an alternative tofluorescence.
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Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
Put forward by
Diplom-Physikerin Eva Rittweger
born in Cologne
thOral examination: 16 December 2009Maximizing far-field optical
microscopy resolution through
selected fluorophore transitions
0
Referees: Prof. Dr. Stefan W. Hell
Prof. Dr. Ju¨rgen WolfrumAbstract:
Stimulated emission depletion (STED) microscopy and related nanoscopy tech-
niques, which utilize a saturable optical transition between a bright and a dark
state, overcome the diffraction barrier by confining one of the states to an area
smaller than the Airy disk. Scanning this area across the specimen yields sub-
diffraction images by registering inseparable fluorescent markers sequentially in
time. Despite the progress made in nanoscopy so far, maximizing the resolu-
tion has been hampered by the efficiency of the utilized optical transition and
the photostability of the fluorophores. Here, the optical transition responsible
for breaking the barrier was studied in order to maximize its efficiency. For a
range of fluorophores (dyes, proteins, quantum dots, color centers) the nature of
the responsible process could be clarified. It was also investigated whether heat
could serve as an imaging contrast to provide an alternative tofluorescence. This
work demonstrates a resolving power of down to 6nm in unprocessed recordings,
corresponding to λ/135, which is to date the highest obtained in far-field optics.
These measurements, which show no sign of photobleaching or blinking, were
performed with diamond color centers using STED and ground state depletion
(GSD) microscopy.
Zusammenfassung:
Die STED- (engl. ”stimulated emission depletion”) Mikroskopie und verwandte
¨Nanoskopie-Methoden, die einen s¨attigbaren optischen Ubergang zwischen einem
hellen und einem dunklen Zustand benutzen, u¨berwinden die Beugungsgrenze,
in dem sie einen dieser Zust¨ande r¨aumlich enger als die Beugungsgrenze ein-
¨schr¨anken. Uberaufgel¨osteBildererh¨altman, indemmandiesenBereichu¨berdie
Probe rastert und so benachbarte fluoreszienende Marker zeitlich sequentiell auf-
nimmt. Trotz vieler Fortschritte in der Nanoskopie war die Au߬osung in der An-
¨wendung beschr¨ankt durch die Effizienz des verwendenten optischen Ubergangs
und die Photostabilit¨at der Fluorophore. In dieser Arbeit wurde der optische
¨ ¨Ubergang, der die Uberwindung der Beugungsgrenze erm¨oglicht, untersucht, um
seineEffizienzzusteigern. Eskonntefu¨reineReihevonFluorophoren(Farbstoffe,
Proteine, Quanten-Dots, Farbzentren)dieFragedeszugrundeliegendenProzesses
gekl¨art werden. Desweiteren wurde untersucht, ob sich W¨arme als alternativer
Bildkontrast zu Fluoreszenz eignet. Es wurde ein Aufl¨osungsverm¨ogen von bis
zu 6nm in Rohdaten, entsprechend λ/135, erreicht, was die zur Zeit h¨ochste
Au߬osung im Fernfeld darstellt. Diese Messungen, die weder Photobleichen oder
Blinken aufweisen, wurden an Diamant-Farbzentren durchgefu¨hrt unter Anwen-
dung der STED- und der GSD- (engl. ”ground state depletion”) Mikroskopie.Contents
1 Introduction 1
2 Mechanisms for fluorescence switching in nanoscopy 5
2.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Pump-Probe Setup . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3.1 Fluorescent markers: dyes, proteins, quantum dots . . . . 8
2.3.2 NV color center in diamond . . . . . . . . . . . . . . . . . 14
3 Heat as an alternative to fluorescence 23
3.1 Temperature change as a contrast mechanism . . . . . . . . . . . 24
3.2 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.3.1 Gold particles . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.3.2 Chromophores . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.3.3 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4 Nanoscopy of diamond color centers 35
4.1 Fundamentals of STED and GSD microscopy. . . . . . . . . . . . 35
4.2 STED microscopy of NV centers . . . . . . . . . . . . . . . . . . . 42
4.2.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.3 GSD microscopy of NV centers . . . . . . . . . . . . . . . . . . . 52
4.3.1 Indirect GSD . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3.2 Direct GSD . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5 Conclusion and outlook 63ii CONTENTS
A Lock in detection 65
B Mn-doped ZnSe quantum dots 67
C Stimulated emission cross section 69Abbreviations
1D one-dimensional
2D two-dimensional
3D three-dimensional
AC alternating current
A.D. anno Domini
B.C. before Christ
CVD chemical vapour deposition
CW continuous wave
Exc excitation
Eff effective
DC direct current
Φ photon flux density in the depletion focusSTEDbΦ photon flux density per laser pulse in the depletion focusSTED
FWHM full width at half maximum
GSD ground state depletion
GSDIM ground state depletion followed by individual molecule return
h confocal point spread functionc
h excitation point spread functionexc
h detection point spread functiondet
I maximum intensity in the depletion focusSTED
I (x) intensity near the depletion focus at position xSTED
k decay rate for spontaneous emission (k = 1/τ )fl fl fl
λ wavelength of the STED beamSTED
λ wavelength of the excitation beamexc
η fluorescence suppression by STED
NA numerical aperture of a lens
NV nitrogen vacancy
−NV negatively charged nitrogen vacancy
0NV neutral nitrogen vacancy
PALM photoactivation localization microscopy
PSF point spread function
RESOLFT reversible saturable optical (fluorescence) transitions
SNR signal to noise ratioiv CONTENTS
SPEM saturated pattern excitation microscopy
STED stimulated emission depletion
STORM stochastic optical reconstruction microscopy
σ stimulated emission cross sectionem
σ excited state absorption cross sectionesa
τ fluorescence lifetimefl
Ti:sapphire Titanium-Sapphire
UV ultraviolet (light)

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