RESOLFT nanoscopy with water-soluble synthetic fluorophores Dissertation for the award of the degree “Doctor rerum naturalium” of the Georg-August-Universität Göttingen within the doctoral program “Physics of Biological and Complex Systems” of the Georg-August University School of Science (GAUSS) submitted by Philipp Johannes Alt from Trier Göttingen 2017 Members of the Thesis Committee Prof. Dr. Stefan W. Hell (Referee) Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Hans-Jürgen Troe (2nd Referee) Institute of Physical Chemistry, Georg-August-Universität Göttingen Dr. Thomas P. Burg Research Group of Biological Micro- and Nanotechnology Max Planck Institute for Biophysical Chemistry, Göttingen Further members of the Examination Board Dr. Melina Schuh Department of Meiosis, Max Planck Institute for Biophysical Chemistry, Göttingen PD Dr. Alexander Egner Department of Optical Nanoscopy, Laser-Laboratorium Göttingen e.V. Prof. Dr. Tim Salditt Research Group for Structure, Dynamics, Assembly and Interaction of Biological Macromolecules, Institute for X-Ray Physics, Georg-August-Universität Göttingen Date of oral examination: 15.12.2017 Abstract Fluorescence microscopy is an important and widely used tool in the life sciences due to its unique ability to observe cellular processes in living specimens with target- specific image contrast. The development of high resolution methods in the far-field has increased its importance further by enabling the visualization of structures fea- turing sizes below the diffraction limit of light. In particular, RESOLFT (reversible saturable optical linear fluorescence transitions) nanoscopy using low light intensities has become a method of choice for live-cell high-resolution fluorescence imaging. RESOLFT nanoscopy requires labels with specialized properties which, to date, have only been observed in reversibly photoswitchable fluorescent proteins (RSFPs). Attempts to extend the palette of RESOLFT labels using synthetic organic fluo- rophores have been limited to proof of concept studies, mostly owing to their in- solubility in water. However, organic fluorophores bear the potential for higher brightness, broader emission and excitation wavelength range as well as higher pho- tostability than RSFPs. In this work the first demonstration of RESOLFT nanoscopy with photoswitch- able diarylethene-based fluorophores in aqueous environments is presented. The photophysical behavior of these novel fluorophores was characterized and compared to RSFPs commonly used for RESOLFT. Furthermore, the fluorophores were suc- cessfully applied to label biological structures in fixed mammalian cells. Imaging based on the RESOLFT concept with these fluorophores yielded two- to threefold improved spatial resolution compared with confocal imaging. The fluorophores pre- sented in this work thus lay the foundation for the further development of synthetic fluorophores for RESOLFT nanoscopy in living specimens. I . Contents Abstract I Contents II List of Abbreviations IV 1 Introduction 1 1.1 Fluorescence ................................2 1.2 Fluorescence microscopes .........................3 1.3 Diffraction limit in far-field light microscopy ..............4 1.4 Diffraction-unlimited microscopy ....................6 1.4.1 Coordinate-targeted switching methods .............6 1.4.2 Coordinate-stochastic switching methods ............8 1.5 RESOLFT nanoscopy with photoswitchable labels ...........9 1.5.1 Requirements for labels used in RESOLFT nanoscopy ..... 11 1.6 Photoswitchable fluorescent proteins .................. 12 1.7 Photoswitchable synthetic fluorophores ................. 14 1.8 Aim of the study ............................. 17 2 Experimental Methods 18 2.1 Absorption and emission measurements in solution .......... 18 2.2 Confocal screening setup ......................... 20 2.3 Confocal and high resolution imaging .................. 24 2.4 Specification of light intensities ..................... 25 2.5 Fluorescence lifetime analysis ...................... 26 2.6 Sample preparation ............................ 26 2.6.1 E. coli expressing fluorescent proteins .............. 26 2.6.2 Preparation of liposomes ..................... 27 2.6.3 Immunofluorescence labeling of cells ............... 28 II . 3 Results 29 3.1 Reversibly photoswitchable fluorescent proteins ............ 29 3.1.1 Typical measurement scheme .................. 29 3.1.2 Different illumination methods .................. 32 3.1.3 Switching fatigue ......................... 33 3.1.4 Switching kinetics at different illumination intensities ..... 34 3.1.5 Fluorescence lifetime ....................... 37 3.2 Synthetic photoswitchable fluorophores without linker ......... 39 3.2.1 Methylated diarylethene ..................... 39 3.2.2 Myo-inositol substituted diarylethenes ............. 44 3.3 Bioconjugateable photoswitchable synthetic fluorophores ....... 47 3.3.1 Photophysical properties in solution ............... 48 3.3.2 Confocal imaging ......................... 50 3.3.3 Fluorescence pump-probe measurements ............ 51 3.3.4 RESOLFT imaging ........................ 57 4 Discussion and Outlook 63 4.1 Reversibly photoswitchable fluorescent proteins ............ 63 4.2 Fluorescent diarylethenes without linker ................ 66 4.3 Bioconjugateable fluorescent diarylethenes ............... 66 4.4 Outlook .................................. 73 A Appendix 75 A.1 Three-state model for RSFPs ...................... 75 A.2 Intensity dependent properties of rsEGFP and rsEGFP2 at low illu- mination intensities ............................ 78 A.3 RESOLFT imaging with EBT2 ..................... 79 A.4 Molecule sizes in fluorescence microscopy ................ 82 Bibliography 83 List of Figures 96 Acknowledgments 99 Curriculum vitae 101 III List of Abbreviations AOM Acousto-optic modulator AOTF Acousto-optic tunable filter APD Avalanche photo-diode BSA Bovine serum albumin CCD Charge-coupled device CF “Closed” form isomer DAE Diarylethene DBS Directional beam splitter DIC Differential interference contrast DOL Degree of labeling DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine EBT 1,2-bis(2-ethyl-6-phenyl-1-benzothiophen-1,1-dioxide-3-yl)- perfluorocyclopentene F(PALM) (Fluorescence) photoactivated localization microscopy FWHM Full width at half maximum GFP Green fluorescent protein GSD Ground state depletion IC Internal conversion ISC Intersystem crossing IRF Instrument response function LED Light emitting diode meDAE Methylated diarylethene mIDAE Myo-inositol substituted diarylethene MMF Multi-mode fiber NA Numerical aperture NHS N -hydroxysuccinimide NI-DAQ National Instruments Data Acquisition (Device) Ni-NTA Nickel-nitrilotriacetic acid IV OF “Open” form isomer PAA polyacrylamide PBS Phosphate buffered saline PFA Paraformaldehyde PMT Photomultiplier tube PSF Point spread function RESOLFT Reversible saturable optical linear fluorescence transitions RET Resonance energy transfer RSFP Reversibly switchable fluorescent protein SBR Signal-to-background ratio SLM Spatial light modulator SMF Polarization-maintaining single-mode fiber SMLM Single molecule localization microscopy SPAD Single-photon avalanche diode STED Stimulated emission depletion STORM Stochastic optical reconstruction microscopy TCSPC Time-correlated single photon counting TIRF Total internal reflection fluorescence TTL Transistor–transistor logic UV Ultraviolet V . 1 Introduction The investigation of processes involved in the evolution of biological life and its functions has been pursued for hundreds of years. The development of magnify- ing optical elements facilitated the exploration of processes which are invisible to the human eye. Although the invention of the microscope itself can be assigned to different individuals in history, it is known that the utilization of these instru- ments revealed the existence of tiny building blocks of life such as blood cells and spermatozoa already in the late 17th century [1]. The detailed optical examination of many biological specimen is hampered without special methods because the structures of interest are translucent, containing small features showing little contrast. It is therefore necessary to increase the contrast between different sample features. On the one hand, the microscope can be tailored to maximize the contrast due to small changes in the local refractive index, e.g. by dark-field illumination, phase contrast or differential interference contrast (DIC) microscopy [2]. On the other hand, the preparation of the specimen remains essential and allows the staining of specific features. For classical transmission microscopy, the image contrast can be enhanced by staining various structures in cells with highly absorptive dyes of different colors. Most recently, the use of fluorophores became one of the most important methods to stain biological samples, because fluorescence and matched color filters provide excellent contrast which allows to detect the signal against an otherwise nearly black background. The possibility to specifically label structures of interest using immunofluorescence or genetically expressed markers in the late 20th century has elevated fluorescence microscopy to an essential tool in the life sciences. Moreover, since light microscopy is not limited to the observation of surfaces, it allows noninvasive imaging of structures and processes inside of living cells and tissues. The basic concepts of fluorescence, fluorescence microscopes, the resolution limit of optical microscopes and the concepts to overcome it will be