STED microscopy with Q-switched microchip lasers [Elektronische Ressource] / presented by Robert R. Kellner

Robert Kellner

Sujets

• Naturwissenschaften

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Dissertation
submittedto the
Combined Facultiesfor the Natural Sciencesand for Mathematics
of the Ruperto­Carola Universityof Heidelberg,Germany
for the degree of
Doctor of NaturalSciences
presented by
Diplom­Physiker Robert R.Kellner
born in Bayreuth
Oral examination: June 20th, 2007STED microscopy
with Q­switched microchiplasers
Referees: Prof. Dr. Stefan W.Hell
Prof. Dr. Josef BilleZusammenfassung
Das Lichtmikroskop ist ein wertvolles wissenschaftliches Instrument in der modernen Biowis-
senschaft,trotzdemwarseinvollesPotentialdurchdiebeugungsbegrenzteAuflösungeingeschrän-
kt. Ein Konzept, welches einen sättigbaren optischen Übergang fluoreszenter Moleküle, nämlich
stimulierteEmission (stimulated emission depletion, STED)ausnutzt,ermöglicht es, diese Begren-
zungzuüberwindenundbieteteinenichtmehrdurchBeugungbegrenzteAuflösung. IndieserAr-
beit wirddieSTED Mikroskopie als Hilfsmittel inder Zellbiologie weiter etabliert, indem Ring-
ähnliche Strukturen des Proteins Bruchpilot in neuromuskulären Verbindungen in Drosophila
Larven,sowiedasAggregationsverhaltendesnikotinischenAcetylcholin-RezeptorsinSäugerzellen
aufgedeckt werden. Darüber hinaus wird die Leistungsfähigkeit von STED Mikroskopen weiter
verbessert, indem das Bleichverhalten fluoreszierender Farbstoffe untersucht wird. Aus diesen
Ergebnissen wird ein neues Beleuchtungsschema für geringeres Photobleichen und erhöhtes Sig-
nal entwickelt. Zum ersten mal wird eine neue Herangehensweise an die STED-Mikroskopie
mit einem gütegeschalteten (Q-switched) Mikrochiplaser, welcher Titan-Saphir basierende Laser-
systeme ersetzt, erfolgreich umgesetzt. Die Anwendbarkeit dieses neuen Ansatzes und eine über
die Beugungsgrenze hinaus erhöhte Auflösung von unter 25nm wird an kolloidalen Nanopar-
tikeln und in biologischen Proben demonstriert. Die Auswahl anwendbare Farbstoffe für die
STED-Mikroskopie wird weiter in den blauen Bereich ausgedehnt und eröffnet dadurch neue
Anwendungsmöglichkeiten.
Abstract
The far-field light microscope is a valuable scientific instrument in modern life sciences, never-
theless, itsfull potentialwas constrained by the diffraction-limitedresolution. Aconcept exploit-
ing a saturable optical transition of fluorescent molecules, namely stimulated emission depletion
(STED), allowed to overcome this restriction and provides diffraction-unlimited resolution in
far-field light microscopy.
In this thesis STED microscopy as a tool in cell biology is further established by revealing the
ring-like structure of the Bruchpilot protein in larval Drosophila neuromuscular junctions and
clustering behavior of the nicotinic acetylcholine receptor in mammalian cells. Moreover, the
performance of STED microscopes is further improved by examining photobleaching behavior
of fluorescent dyes and presenting a new illumination scheme for reduced photobleaching and
increased signal. For the first time a new approach to STED microscopy with a Q-switched
microchip laser, replacing a Ti:Sapphire-based laser system, was successfully implemented. The
applicabilityof this approach and an increase in resolution beyond the diffraction limit to below
25nm is demonstrated with colloidal nanoparticles and in biological samples. The range of ap-
plicable dyes for STED microscopy is further extended into the blue regime, widening the field
of possibleapplications.Contents
1 Introduction 1
1.1 Microscopy incell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Modernlight microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.1 Specialimaging modes . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Novel light microscopy techniques . . . . . . . . . . . . . . . . . . . . 2
1.3 Towardsnanoscale resolution infar-field light microscopy . . . . . . . . . . . . 3
1.3.1 Theresolution issue . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3.2 Overcoming the barrier . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Advances inSTED microscopy 8
2.1 TheSTED Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2 PSFengineering for STEDmicroscopy . . . . . . . . . . . . . . . . . . . . . . 10
2.3 Spectralcharacterizationof suitableSTED-dyes . . . . . . . . . . . . . . . . . . 11
2.4 Examining photobleaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.4.1 Signalincrease instandard confocal microscopy . . . . . . . . . . . . . 13
2.4.2 Predominant bleachingpathwaysinSTED microscopy . . . . . . . . . 16
2.5 STEDapplicationsincellbiology . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.5.1 STEDmicroscopy reveals ring-likestructuresinDrosophilaNMJ . . . . 19
2.5.2 STEDmicroscopy for investigationof acetylcholinereceptors . . . . . . 22
3 STED microscopy withQ­switched microchip lasers 28
3.1 Theshift toQ-switchedmicrochip lasers . . . . . . . . . . . . . . . . . . . . . 28
3.1.1 Anew approachwithQ-switched microchip lasers . . . . . . . . . . . . 28
3.1.2 BluedyesforSTED microscopy . . . . . . . . . . . . . . . . . . . . . . 29
3.2 TheSTED setupusing Q-switchedmicrochip lasers . . . . . . . . . . . . . . . 30
3.2.1 Pulsetimingand beam shaping . . . . . . . . . . . . . . . . . . . . . . 31
3.2.2 Increasing thespeed of Q-switched microchip laserSTEDmicroscopy . 32
3.2.3 Thefinal setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3 Sub-diffractionresolution demonstrated . . . . . . . . . . . . . . . . . . . . . . 34
4 Conclusion and outlook 37
Bibliography 39
iiContents iii
A Appendix 46
A.1 Imaging acetylcholinereceptors . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A.1.1 Cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A.1.2 Preparationof single plasmamembrane sheets . . . . . . . . . . . . . . 46
A.1.3 Cholesterol depletionof cellsand single plasmamebrane sheets . . . . . 46
A.2 Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A.2.1 PtK2cell preparationforbleachingexperiments . . . . . . . . . . . . . 46
A.2.2 Mowiol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
A.2.3 Preparationof fluorescent nanoparticles . . . . . . . . . . . . . . . . . . 47
List of publications 49Abbreviations
2D two-dimensional
3D three-dimensional
AChR acetylcholinereceptor
AFM atomic force microscope
APD avalanche photodiode
BRP Bruchpilot
CARS coherent anti-StokesRaman-scattering
CSR complete spatialrandomness
CDx methyl- -cyclodextrin
CI confidence interval
CLSM confocal laserscanning microscope
FLIM fluorescence lifetimeimaging
FRAP fluorescence recovery afterphotobleaching
FRET Förster resonance energy transfer
FWHM full-widthhalf-maximum
NMJ neuromuscular junction
OPA opticalparametric amplifier
OPO opticalparametric oscillator
PBS polarizingbeam-splitter
PCF photonic crystalfiber
PSF point spread function
PVA poly(vinylalcohol)
RegA regenerative amplifier
RESOLFT reversible saturable optical(fluorescent) transition
SEM scanning electron microscope
SNOM scanning near-field opticalmicroscope
SLM spatiallight modulator
STED stimulated emission depletion
STM scanning tunnelingmicroscope
TEM transmission electron microscope
Ti:Sapphire titanium-sapphire
VLP virus-likeparticle
iv
1 Introduction
1.1 Microscopyin cell biology
When Robert Hooke used a microscope in 1664 to observe “minute objects” [1] he used the
focused light of an oil lamp to illuminate his samples. Differences in reflectivity and in the
refractive indexwithinthe investigated structureallowed himtodistinguishsmall features. Inhis
work,Hookecoinedtheterm“cell”andpresentedthefirstgraphicalrepresentationofmicroscopic
objects.
Over the next centuries, light microscopes were improved and have become an important tool
forscientistsfromdifferentfields. In1873,ErnstAbbediscoveredthattheresolutionofafar-field
lightmicroscopeisessentiallylimitedbydiffractiontoslightlylessthanhalfthewavelengthofthe
imaging light [2].
Tocircumventthisbarrier,newtypesofmicroscopesthatdonotutilizelighttogenerateimages
have emerged over the last decades. The development of geometric electron optics[3, 4] and the
transmission electron microscope (TEM) in 1932 [5] opened up new possibilities, especially in
cell biology. The scanning electron microscope (SEM) was derived just a few years later. Modern
TEMs can image with a resolution of close to or below 0.1nm [6]. Unfortunately, the high en-
ergy electron beams used in these microscopes are destructive for biological material. Moreover,
the samples under investigation must be fixed, embedded in epoxy or frozen and imaged un-
der vacuum conditions. These restrictions make electron microscopy incompatible with live-cell
imaging.
Scanning probe microscopy techniques, like atomic force microscopy (AFM), scanning tun-
neling microscopy (STM), and scanning near-field optical microscopy (SNOM) also provide the
possibility to image samples at the atomic scale. Especially AFM and SNOM are able to image
small detailsof biologicalspecimen and have proven valuable forcellbiology [7]. However, scan-
ning probe microscopy is inherently surface bound and therefore incapable of thr