Cover Page The handle http://hdl.handle.net/1887/37175 holds various files of this Leiden University dissertation

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Cover Page

The handle http://hdl.handle.net/1887/37175 holds various files of this Leiden University dissertation

Author: Harkes, Rolf

Title: Quantitative super-resolution microscopy

Issue Date: 2016-01-13

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Quantitative Super-Resolution Microscopy

PROEFSCHRIFT

Ter verkrijging van

de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. C.J.J.M. Stolker,

volgens besluit van het College voor Promoties te verdedigen op woensdag 13 januari 2016

klokke 11.15 uur

door

Rolf Harkes geboren te Wageningen

in 1986

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Promotor: Prof. dr. T. Schmidt

Promotiecommissie: Dr. G.A. Blab (Universiteit Utrecht) Prof. dr. A. Diaspro (IIT, Genova, Italië) Prof. dr. V. Subramaniam (Vrije Universiteit) Prof. dr. E.R. Eliel

Prof. dr. A.J. Koster Dr. ir. S.J.T. van Noort Prof. dr. M.A.G.J. Orrit

© Rolf Harkes. All rights reserved.

Cover front: 3D dSTORM image of GFAP in Astrocyte Cover back: Optical setup used for super-resolution imaging Casimir PhD Series, Delft-Leiden, 2015-35

ISBN 978-90-8593-241-3

An electronic version of this thesis can be found at https://openaccess.leidenuniv.nl

Het onderzoek beschreven in dit proefschrift is onderdeel van het wetenschappelijke

programma van de Stichting voor Fundamenteel Onderzoek der Materie (FOM), die

financieel wordt gesteund door de Nederlandse organisatie voor Wetenschappelijk

Onderzoek (NWO).

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“The only acceptable point of view appears to be the one that recognizes both sides of reality: the quantitative and the qualitative.”

Wolfgang Pauli -Writings on Physics and Philosophy-

Dedicated to Sietske Froukje Harkes-Idzinga

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TABLE OF CONTENT

1. INTRODUCTION INTO SUPER-RESOLUTION

MICROSCOPY 1

1.1 Theory of microscopy 2

1.2 Super-resolution microscopy techniques 4

1.2.1 Structured illumination ... 4

1.2.2 Near-field scanning microscopy ... 5

1.2.3 Fluorescence ... 6

1.2.4 Stimulated emission depletion ... 6

1.3 Single molecule fluorescence 8 1.3.1 Photoactivatable fluorescent proteins ... 8

1.3.2 PALM STORM and fPALM ... 9

1.3.3 dSTORM ... 10

1.3.4 3D SMLM ... 11

1.4 Comparison between imaging techniques 14 1.5 Quantification of single molecule data 15 1.5.1 Nyquist-Shannon sampling theorem ... 15

1.5.2 Image construction ... 18

1.5.3 Stoichiometry and multiple detections ... 19

1.6 Outline of this thesis 21 1.7 References 23 2. SINGLE MOLECULE STUDY OF RAS MEMBRANE DOMAINS REVEALS DYNAMIC BEHAVIOR 25 2.1 Introduction 26 2.2 Materials and methods 28 2.2.1 Microscope ... 28

2.2.2 Correction for double detections ... 28

2.2.3 Cell culture and transfection ... 29

2.2.4 Analysis software ... 29

2.2.5 Ripley ... 29

2.2.6 Bootstrapping and automatic selection ... 30

2.2.7 Simulation of cluster diffusion ... 30

2.3 Results 31 2.3.1 Imaging of H-CAAX in fixed COS1 cells ... 31

2.3.2 Imaging of H-CAAX in living COS1 cells ... 35

2.4 Discussion 42

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2.5 Outlook 43

2.6 References 45

2.7 Supplementary figures 47

3. 3D DIFFUSION MEASUREMENTS OF

THE GLUCOCORTICOID RECEPTOR 49

3.1 Introduction 49

3.2 Methods 51

3.2.1 Cell culture ... 51

3.2.2 Single molecule imaging ... 51

3.2.3 Particle image correlation spectroscopy (PICS) analysis ... 52

3.2.4 Depth of field calibration ... 53

3.3 Results 54 3.3.1 Analytical solution for correction of fraction size in 3D diffusion with limited detection volume ... 54

3.3.2 Validation of the correction by simulations ... 57

3.3.3 Validation of the correction using experimental data ... 58

3.4 Conclusion 62 3.5 References 64 4. DIRECT OBSERVATION OF Α-SYNUCLEIN AMYLOID AGGREGATES 67 4.1 Introduction 68 4.2 Materials and methods 70 4.2.1 Preparation of labeled α syn fibrillar seeds ... 70

4.2.2 Cell culture ... 70

4.2.3 Atomic force microscopy (AFM) ... 70

4.2.4 Co-localization experiments with lysosomes ... 71

4.2.5 dSTORM experiments and data analysis... 71

4.3 Results and discussion 74 4.3.1 Super-resolution imaging of in vitro α syn fibrils ... 74

4.3.2 Internalization of extracellular α syn fibrils into neuronal cells ... 77

4.4 References 82

4.5 Supplementary figures 84

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5. FORCE SENSING AND QUANTITATIVE dSTORM ON SIGNAL TRANSDUCTION PROTEINS 85

5.1 Introduction 86

5.2 Materials and methods 89

5.2.1 Cell culture ... 89

5.2.2 Sample preparation ... 89

5.2.3 dSTORM ... 90

5.2.4 Image analysis ... 91

5.3 Results 92 5.3.1 Analysis framework ... 92

5.3.2 Counting molecules in focal adhesions ... 98

5.4 Discussion 102 5.5 Outlook 102 5.6 References 103 5.7 Supplementary materials 105 5.7.1 Relation between variance and squared mean ... 105

5.7.2 Simulation for a combined statistics with secondary antibody labeling... 105