Dissertation zur Erlangung des Doktorgrades der Fakult¨atf¨urChemie und Pharmazie der Ludwig-Maximilians-Universit¨atM¨unchen Assembly and optimization of a super-resolution STORM microscope for nanoscopic imaging of biological structures Jens Prescher aus M¨unchen, Deutschland 2016 Erkl¨arung Diese Dissertation wurde im Sinne von x7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Don C. Lamb, PhD betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenst¨andigund ohne unerlaubte Hilfe erarbeitet. M¨unchen, den 13. Januar 2016 ............................................ Dissertation eingereicht am 15. Januar 2016 1. Gutachter Prof. Don C. Lamb, PhD 2. Gutachter Prof. Dr. Christoph Br¨auchle M¨undliche Pr¨ufungam 18. Februar 2016 Contents Abstract v 1 Introduction 1 2 Theory of super-resolution fluorescence microscopy 5 2.1 Principles of fluorescence . 5 2.2 Fluorescence microscopy and optical resolution . 8 2.2.1 Principle of fluorescence microscopy . 9 2.2.2 Resolution in optical microscopy . 10 2.2.3 Total Internal Reflection Fluorescence Microscopy . 14 2.3 Super-resolution fluorescence microscopy . 17 2.3.1 Localization-based super-resolution microscopy . 18 2.3.1.1 Photoactivated Localization Microscopy (PALM) . 21 2.3.1.2 Stochastic Optical Reconstruction Microscopy (STORM) . 22 2.3.1.3 Direct Stochastic Optical Reconstruction Microscopy (dSTORM) 23 2.3.2 Determination of resolution in STORM and PALM . 24 2.3.3 Three-dimensional localization-based super-resolution imaging . 25 2.3.4 Overview over other optical super-resolution methods . 27 3 Experimental setup and analysis methods for STORM 29 3.1 Experimental setup . 29 3.1.1 Instrumentation of the STORM setup . 29 3.1.2 Implementation and tuning of a perfect focus system for increased focus stability . 32 3.1.3 EMCCD camera characterization and mapping . 35 3.1.3.1 Count to photon conversion for EMCCD cameras . 35 3.1.3.2 Camera mapping . 37 3.2 STORM data acquisition and analysis protocol . 39 3.2.1 Test systems . 39 3.2.2 Data acquisition . 40 3.2.3 Fluorescent emitter localization . 42 3.2.3.1 Gaussian least squares fitting . 43 3.2.3.2 Maximum likelihood estimation . 47 3.2.4 Image rendering . 49 3.2.5 Correction of lateral drift . 51 3.3 Calculation of image resolution . 53 3.4 Calculation of cluster sizes in super-resolution images . 56 3.5 2D dSTORM imaging of HeLa cell microtubules . 59 3.6 Implementation of three-dimensional STORM and dSTORM . 61 3.6.1 Astigmatism calibration for 3D STORM and dSTORM imaging . 61 3.6.2 3D dSTORM imaging of test samples and data analysis . 64 3.7 Multi-color dSTORM . 67 3.8 Summary & Outlook . 69 i Contents 4 Super-resolution imaging of ESCRT proteins at HIV-1 assembly sites 71 4.1 Introduction . 71 4.1.1 Acquired immunodeficiency syndrome (AIDS) and HIV . 71 4.1.2 HIV-1 life cycle . 73 4.1.3 ESCRT-mediated budding of nascent HIV-1 particles . 75 4.2 Experimental design . 78 4.3 Results . 80 4.3.1 Characterization of HIV-1 assembly sites using PALM . 80 4.3.2 Verification of efficient immunostaining inside nascent HIV-1 buds . 82 4.3.3 Characterization of ESCRT proteins and ALIX at HIV-1 budding sites . 83 4.3.3.1 Super-resolution imaging of endogenous of Tsg101 . 85 4.3.3.2 Distribution of membrane-bound ALIX and association to HIV-1 budding . 87 4.3.3.3 CHMP4B-HA lattices colocalizing with HIV-1 assembly sites . 90 4.3.3.4 Super-resolution imaging of CHMP2A lattices . 94 4.3.4 Super-resolution imaging of YFP-tagged Tsg101 at HIV-1 assembly sites . 96 4.3.5 Effect of a dominant-negative VPS4 mutant on ESCRT structures . 99 4.3.6 Relative orientation of ESCRT with respect to HIV-1 . 101 4.3.6.1 Dual-color super-resolution imaging of ESCRT and HIV-1 bud- ding sites . 101 4.3.6.2 Determination of relative orientation by overlaying widefield and super-resolution images . 102 4.4 Discussion . 103 5 Super-resolution imaging of lamellipodia structures 109 5.1 Introduction . 109 5.1.1 Actin . 109 5.1.2 Cellular migration and lamellipodia . 110 5.1.3 Micro-environment influence on cell motility . 112 5.2 Experimental design . 113 5.3 Results . 114 5.4 Discussion . 117 6 Conclusion 119 A Materials and Methods 121 A.1 STORM sample preparation protocols . 121 A.1.1 STORM imaging buffer . 121 A.1.2 Antibody labeling . 121 A.1.3 STORM bead sample preparation . 122 A.1.4 3D calibration sample preparation . 122 A.1.5 Microtubules sample preparation protocol . 122 A.2 Sample preparation and analysis of super-resolution imaging of ESCRT proteins 123 A.3 Sample preparation protocols for super-resolution imaging of endothelial lamel- lipodia . 125 A.4 Optimization algorithms . 125 A.4.1 Levenberg-Marquardt algorithm . 126 A.4.2 Downhill Simplex algorithm . 126 B Abbreviations 129 ii Contents List of Figures 131 List of Tables 132 Bibliography 133 Acknowledgments 155 iii iv Abstract Fluorescence microscopy is a widely used technique for imaging of biological structures due to its noninvasiveness although resolution of conventional fluorescence microscopes is limited to about 200{300 nm due to the diffraction limit of light. Super-resolution fluorescence microscopy offers an extension of the original method that allows optical imaging below the diffraction limit. In this thesis, a microscope for localization-based super-resolution fluorescence microscopy techniques such as Stochastic Optical Reconstruction Microscopy (STORM) or Photoactivated Localization Microscopy (PALM) was established. An epifluorescence microscope was built for this purpose that provides both widefield and Total Internal Reflection Fluorescence (TIRF) excitation modalities and focus was put on the special requirements of localization-based super- resolution methods. This included a high mechanical and optical stability realized by a compact design and implementation of a home-built perfect focus system. The setup was further designed to allow both two- and three-dimensional imaging. The work also included both the development of a setup control software and a software for the analysis of the required data. Different analysis methods and parameters were tested on simulated data before the performance of the microscope was demonstrated in two and three dimensions at appropriate test samples such as the cellular microtubule network. These experiments showed the capability of super-resolution microscopy to reveal underlying structures that cannot be resolved by conventional fluorescence microscopy. Resolutions could be achieved down to approximately 30 nm in the lateral and 115 nm in the axial dimension. Subsequently, the established method was applied to two biological systems. The first is a study of the budding of the human immunodeficiency virus type 1 (HIV-1) from the host cell. In this step of the viral reproduction cycle, the virus hijacks the cellular endosomal sorting complex required for transport (ESCRT) machinery to achieve membrane fission. ESCRT consists of the subcomplexes ESCRT-0, -I, -II and -III and additional related proteins, from which HIV-1 recruits certain components. The fission process is initiated by the HIV-1 Gag protein, which recruits the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX to the virus assembly site. Subsequently, ESCRT-III proteins CHMP4 and CHMP2 form transient lattices at the membrane, which are actively involved in membrane fission. However, the actual geometry of the ESCRT machinery assembling at the HIV-1 budding site that is driving the fission process is still not fully understood. Different models proposed either constriction of the budding neck by lattices surrounding the neck, by ESCRT structures within the neck or within the bud itself. A problematic aspect in previous studies was the usage of modified, tagged versions of the involved proteins for visualization. In this study, super-resolution microscopy was therefore applied to endogenous Tsg101, ALIX and CHMP2 isoform CHMP2A and to a version of CHMP4 isoform CHMP4B with a small HA-tag to elucidate the size and the distribution of the structure relative to the HIV-1 assembly sites. ESCRT structures colocalizing with HIV-1 exhibited closed, circular structures with an average size restricted to 45 and 60 nm in diameter. This size was significantly smaller than found for HIV-1 assembly sites and the constriction of the size, which was not observed for non-colocalizing ESCRT structures at the cell membrane, ruled out an external restriction model. Super-resolution imaging of ALIX often revealed an additional cloud-like structure of individual molecules surrounding the central clusters. This was attributed to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Together with v Abstract experiments that confirmed the non-physiological behavior of tagged Tsg101 and a relative orientation of ESCRT clusters towards the edge of the assembly site, the results strongly point toward a within-neck model. A second project focused on the influence of external constriction on cell migration. The latter plays an important role in various processes in the human body ranging from wound healing to metastasis formation by cancer cells. Migration is driven by the lamellipodium, which is a meshwork of fine actin filaments that drive membrane protrusion. Endothelial cells were grown on micropatterns that confined the freedom of movement of the cells. Three-dimensional super-resolution imaging revealed that the lamellipodia of these cells showed a much broader axial extension than was the case for control cells that grew without confinement of migration. The different organization of the actin filament network showed a clear effect of environmental conditions on cellular migration. Overall, it was possible to build a super-resolution fluorescence microscope over the course of this study and establish the required analysis methods to allow STORM and PALM imaging below the diffraction limit of light.