High-Resolution Fluorescence Microscopy with Photoswitchable Fluorescent Proteins Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades ”Doctor rerum naturalium” der Georg-August-Universitat¨ Gottingen¨ vorgelegt von Hannes Bock aus Hamburg Gottingen,¨ 2008 . D 7 Referent: Prof. Dr. M. Munzenberg¨ Koreferent: Prof. Dr. S. W. Hell Tag der mundlichen¨ Prufung:¨ High-Resolution Fluorescence Microscopy with Photoswitchable Fluorescent Proteins Summary The diffraction limit in far-field fluorescence microscopy can be overcome by photoswitch- ing the marker molecules between a fluorescent and a non-fluorescent state. Photoswitching enables the successive generation and readout of nano-sized fluorescent areas in the sample. Subsequently, these areas can be assembled to form a super-resolution image of the speci- men. For the first time in this work, one and the same molecular switching mechanism of a reversibly switchable fluorescent protein (RSFP) was used to realize two alternative varia- tions of this general nanoscopy principle: one based on the targeted switching of ensembles of molecules and the other based on the stochastic switching of single emitters. RSFPs are of particular interest for these techniques because they allow the non-invasive study of pro- cesses in living cells and can be efficiently switched using low light intensities, thus minimiz- ing photo stress and possible photo-induced damage of the cell. Furthermore, the protein’s photoswitching properties can be improved via targeted mutagenesis. In the case of both nanoscopy techniques presented here, image acquisition could be significantly improved and accelerated as compared to related approaches. For the first time, two-color far-field fluores- cence nanoscopy based on the photoswitching of single emitters was successfully realized in biological samples. Zusammenfassung Die Beugungsgrenze in der Fluoreszenzfernfeldmikroskopie kann durch lichtinduziertes Schal- ten der verwendeten Farbstoffmolekule¨ zwischen einem fluoreszenten und einem nichtflu- oreszenten Zustand uberwunden¨ werden. Dazu werden in der Probe durch Fotoschalten nacheinander fluoreszente Bereiche erzeugt und ausgelesen, deren Ausdehnung nicht durch die Wellenlange¨ des Lichtes begrenzt ist. Durch das nachtragliche¨ Zusammensetzen dieser nanoskopischen Bereiche entsteht ein hochaufgelostes¨ Bild der Probe. In dieser Arbeit konnten zum ersten Mal mit ein und demselben reversibel schaltbaren Fluoreszentprotein (RSFP) zwei verschiedene Umsetzungen dieses allgemeinen Prinzips realisiert werden, die zum einen auf dem gezielten Schalten von Ensemblen von Molekulen¨ und zum anderen auf dem zufalligen¨ Schalten einzelner Molekule¨ beruhen. RSFPs sind fur¨ diese Verfahren von großer Bedeutung: Sie konnen¨ Prozesse in lebenden Zellen nichtinvasiv sichtbar machen; sie konnen¨ bei niedrigen Lichtintensitaten¨ effektiv geschaltet werden, was die Einwirkung auf die Zelle minimiert; und sie konnen¨ in ihren Eigenschaften als Fotoschalter durch Muta- genese gezielt verbessert werden. Beide der hier vorgestellten Nanoskopieverfahren stellen in ihrer Umsetzung durch eine erhebliche Verminderung der Bildaufnahmezeit eine deut- liche Verbesserung im Vergleich zu fruheren¨ Ansatzen¨ dar. Zusatzlich¨ wurde zum ersten Mal einzelmolekulbasierte¨ Zwei-Farben-Nanoskopie in biologischen Proben realisiert. 4 Contents Introduction 6 1 Resolution in Optical Microscopy 9 1.1 The Diffraction Limit . .9 1.2 Sub-Diffraction Techniques . 11 1.2.1 RESOLFT Microscopy . 12 1.2.2 Single Molecule Switching Microscopy . 16 2 The RSFP rsFastLime 18 2.1 Reversible Switching at the Ensemble- and Single Molecule Level . 20 2.2 RESOLFT Microscopy with rsFastLime . 27 2.2.1 Imaging with rsFastLime . 33 2.3 Detailed Ensemble Photophysics of rsFastLime . 47 2.4 Single Molecule Switching (SMS) Microscopy with rsFastLime . 54 2.5 Summary of RESOLFT- and SMS Microscopy with rsFastLime . 63 3 Two-Color Single Molecule Switching Microscopy 68 3.1 Dark State Shelving of Cy5 . 68 3.2 Two-color Imaging of Biological Samples . 71 4 The RSFP ASFP595 75 4.1 Photoswitching Properties . 76 4.2 Super-Resolution RESOLFT Imaging . 77 5 Conclusion 80 Bibliography 82 A Appendix 92 A.1 Expressions for the Resolving Power . 92 A.2 Photophysical Model of rsFastLime . 93 A.3 Staining of Tubulin in PtK2 Cells . 94 A.4 List of Scientific Contributions . 95 B Danksagung 96 5 Introduction By pushing open the door to a previously unknown world, the invention of the far-field light microscope in the 16. and 17. century truly marked a milestone in the history of science. In the centuries to follow it lead scientists to major new insights ranging from the discovery of the cell as the basic structural and functional unit of living tissue to the observation of statis- tical molecular motion by R. Brown (now known as Brownian motion), to name only a few. Since then the far-field light microscope has found a wide scope of applications, particularly in the biomedical sciences. Following the seminal work of Ernst Abbe in the second half of the nineteenth century [1] it became commonly accepted that the resolving power of the light microscope was limited by diffraction to about half the optical wavelength, i.e. typically to ≈ 200nm in the lateral and ≈500nm in the axial direction. Subsequently, the twentieth century brought about the devel- opment of imaging technologies capable of resolving structures on the atomic scale. E.g., the transmission electron microscope (TEM)[2] makes use of the short de Broglie wavelength of accelerated electrons (≈100keV). In scanning tunneling microscopy (STM)[3] a conducting tip measures the tunneling current as the tip is moved over the surface of the (conducting) sample. In an atomic force microscope (AFM)[4], on the other hand, it is the interaction be- tween the scanning tip and the surface atoms that provides the information about the surface structure. All of these methods have spurred scientific breakthroughs. However, although they provide a more than ≈ 1000-fold improved resolution over optical microscopy, they suffer from a number of limitations when it comes to the imaging of biological specimens. Namely, they are mostly restricted to the study of the sample surface or necessitate cutting it into thin slices; also, they generally require a sample preparation protocol which is not compatible with the imaging of living objects, including e.g. de-hydration, evacuation or cooling of the specimen to cryogenic temperatures. Not being subjected to any of the aforementioned restraints, far-field fluorescence microscopy has emerged as one of the most powerful and versatile tools in the life sciences in the course of the past decades. The methodology relies on the specific staining of target proteins or or- ganelles with fluorescent labels in combination with excitation of the fluorescent marker by a light source of a suitable wavelength, typically operating in the visible spectrum between 450 and 700nm. Thus, it bears the significant advantage of permitting true three dimensional imaging of translucent specimens. Even more importantly, far-field fluorescence microscopy enables the imaging of living cells. What adds to its great capacity is a highly developed la- beling technology that allows to mark defined proteins or cellular compartments with high efficiency and specificity. In this work, the final remaining drawback of far-field fluorescence microscopy is addressed, namely its limited resolving power which until recently prevented the study of nanoscale 8 structures and processes occurring in living cells. However, in the last decade a number of nanoscopy methodologies based on the photoswitching of the fluorescent labels have emerged which are capable of breaking the diffraction limit of far-field light microscopy formulated by Abbe about a century and a half ago [5]. It can be anticipated that in the near future further progress in imaging technology in connection with the development and refinement of new switchable fluorescent probes will significantly enhance our knowledge about fundamental cellular processes. 1 Resolution in Optical Microscopy 1.1 The Diffraction Limit As a noninvasive imaging technique which allows the observation of living specimens far- field fluorescence microscopy has gained great importance in recent decades. However, it suffers from its limited resolution. The resolution of an imaging apparatus is defined as the minimum distance at which two similar objects can still be discerned. Consequently, the device is not capable of revealing the details of structures which are smaller than this minimum resolvable distance. E. Abbe was the first to point out that in a far-field light microscope diffraction fundamentally limits the resolution ability to a scale which is set by the optical wavelength [1]. Specifically, he stated that the lateral resolution ∆r and the axial resolution ∆z of such an instrument can be expressed as λ 2λ ∆r ≈ and ∆z ≈ ; (1.1) 2n sin(α) n sin2(α) where λ is the wavelength of the light used in the image formation process, n is the index of refraction of the surrounding medium and α the semi aperture angle of the objective lens. The quantity NA = n sin(α) is commonly referred to as the numerical aperture of the system. Nowadays, high aperture oil-immersion objective lenses feature numerical apertures of NA ≈1.4. According to Abbe, (1.1) denotes both the extension of the far-field optical image of a point-like source and the width of the smallest possible focus to which a beam of light can be brought using the