Title Promoter Affiliations Abstract "Multidisciplinary installation of a polyvalent microscope for transmitted light, phase contrast and fluorescence microscopy" "Paul Lambrechts" "Biomaterials - BIOMAT, Department of Imaging & Pathology, Periodontology & Oral Microbiology, Orthodontics, Population Studies in Oral Health" "Several research lines within the Department of Oral Health Sciences are using light microscopy in several configurations (transmitted light, phase contrast, fluorescence) and they search externally for the observation of histological and microbiological samples and for image digitalisation. However, the screening of slices and samples needs to be performed in close proximity to the place where the samples are prepared, coloured and observed. The main goal is to realise the multidisciplinary installation of a polyvalent microscope for transmitted light, phase contrast and fluorescence microscopy. In this way soft tissue and hard tissue histology becomes possible, together with microbiological research within a research landscape where several research clusters are concentrated.We choose for a Leica DM 3000 LED microscope with polyvalent camera and LAS software, coupled to a powerful computer with a high resolution screen." "Novel microscope designs for fast and highly adaptable fluorescence microscopy." "Wim Vandenberg" "Biochemistry, Molecular and Structural Biology" "Due to its high sensitivity and specificity, fluorescence microscopy has been a workhorse technique in (bio-)chemical and medical research for several decades. In recent years, thanks to advances in labeling chemistry, the problems to which this technique can be applied have been strongly diversified. However, the application of these new technologies in 3D rather than 2D microscopy has been limited, at least in part due to a lack of adaptable, robust, sensitive, fast & affordable instruments. Within this project we will tackle this problem trough the development of state-of-the-art microscopes built on a novel optical designs." "The development, screening and characterization of novel fluorescent probes for applications in fluorescence microscopy with nanometer resolution." "Johan Hofkens" "Molecular Imaging and Photonics, Biochemistry, Molecular and Structural Biology" "The applicant proposes a project for the systematic development, screening, spectroscopic and photophysical characterization of new fluorescent probes for diffraction-unlimited fluorescence microscopy. Fluorescence microscopy is unique in its possibility for selective, minimally invasive measurements in living cells and tissues, and is a standard tool in any biophysical research lab. By default this technique is limited to a spatial resolution of several hundred nanometers by the diffraction of light, which is very large in relation to the characteristic nanometer-length scales on which structure and dynamic processes occur in living matter. Recently however, new techniques, including the applicant, a diffraction-unlimited resolution possible. These techniques are based on using specially designed fluorophores in combination with the stochastic nature of single-molecule processes. Currently, the performance of these techniques is limited mainly by the absence of fluorophores with optimum physical properties. The project consists of the design, characterization and application of new fluorophores with improved properties." "In situ investigation of biochemical and nanocolloidal dynamics using fast optical nanoscopy: Video rate high resolution fluorescence microscopy in material science and biochemistry." "Johan Hofkens" "Molecular Imaging and Photonics" "Nanomaterials are all around us. They are widely used as they show some extraordinary properties as catalysts (showing unexpected reactivity or needing less starting material), as additives to change properties of bulk materials (exhibiting luminescent properties like in quantum dots, or altering wetting behavior for self cleaning effect), biomedical applications for target drug delivery, potential investigation tool or new treatment procedure. Most of these applications seem futuristic, yet nanomaterials are present in many household objects such as sunblock (sun burn protection), toothpaste (for improved cleansing and protection), in fridges (antimicrobial) and in computers (for miniaturization). To study their behavior, interaction and influence one needs to be able to visualize these materials, and their dynamics, at the real size. For years biologists have used optical microscopy to study microscopic life, such as bacteria and viruses. Hence optical microscopy has proven itself as a convenient tool for studying microscale materials. Especially fluorescence microscopy, such as confocal microscopy, has been shown to be extremely useful in investigating dynamics of (sub)micron particles, with limiting disturbance of the sample. Nowadays, improvements in the field of optical microscopy, such as STED, allow even to obtain tens of nanometers in resolution. These techniques are classified as super-resolution fluorescence microscopy techniques or optical nanoscopy techniques. As to control the location of the material of interest, droplets can be used. Droplets are a commonly encountered, three phase system (of liquid, gas and solid) and are or a side effect or a goal in many industrial and academic research.Droplets are an easy way to assemble nano and biomaterials, in a controlled way.  This thesis aims to show the usefulness of fluorescence microscopy for studying sub-micron particles, of any origin, in a controllable model system: i.e. evaporating droplets. The studies in this PhD deal with droplet induced substrate assembly of nanoparticles, microorganisms or DNA (a biomolecule well-known for carrying genetic information). Additional, supplementary information about the results is put at the end of this dissertation, in the respective appendices. The well-known coffee ring effect was exploited. This is a flow inside a droplet that is present when it is pinned to a substrate and creating, upon drying, ring like deposits of the present suspended matter. Drops containing fluorescent bacteria and colloids, show this coffee ring flow. Upon addition of surfactants this flow profile changes, and subsequently also the final deposition. Remarkably, we see that bacteria that produce surfactants themselves show comparable flows and altered deposits.  A follow-up study with a higher surfactant concentration showed a macroscopic repetitive assembly of the colloids is reached, along the droplet edge, and this in the shape of a flower. In a third study, droplets containing fluorescent DNA were used to deposit linear fragments of DNA on a substrate. Tuning the substrate influences the efficiency of DNA retention and stretching. Further optimization, by droplet pulling, allowed efficient DNA linearization and binding, even at extremely low concentrations of DNA. DNA can be linearly stretched and can reach lengths of micrometers. Yet DNA is 1D nanoscale system, as the base pairs only are a few nanometers wide. In the last study, tSTED (an extension of the high resolution STED technique) is used to investigate DNA assemblies on dry substrates. Varying buffer parameters allowed alternating DNA deposits, visual via tSTED in high resolution. Additional experiments showed that the DNA probe, YOYO-1 could interact with itself and deteriorate the image quality of STED. tSTED imaging allows us to overcome this deterioration and the life time information provides an additional resolution improvement.This laserscanning tSTED platform will allow studying DNA stretching by droplets, in high spatial and temporal resolution, where other techniques would fail." "Super resolution fluorescence microscopy of biomolecular interactions with photochromic biosensors and complementation." "Johan Hofkens" "Biochemistry, Molecular and Structural Biology, Molecular Imaging and Photonics" "Fluorescence microscopy techniques are indispensable research tools that have enabled live cell imaging with low invasiveness, high sensitivity, and high spatiotemporal resolution. As a result, fluorescence microscopy has revolutionized our understanding of the inner workings of single cells and whole organisms. An integral part of the imaging performance is the fluorescent labels, as the quality of the labels influences the quality of the data. Therefore, researchers have continuously focused on the development of new and improved fluorophores. This is also true for fluorescent proteins (FPs), the genetically encoded labels that allow highly specific targeting, labeling and tracking in living cells.In the last decades, the toolbox of available FP variants has been expanded substantially, through the discovery of new FPs in various marine organisms, and by the engineering of existing FPs. An intriguing addition to this toolbox are the phototransformable FPs, which can undergo a photoinduced transformation that changes their spectral properties. These photophysically “smart” FPs are often relied upon for advanced imaging schemes, such as super-resolution fluorescence microscopy, single particle tracking or dynamic labeling. One example of phototransformable FPs are the reversibly switchable, or photochromic, FPs. Another impressive feat of FP technology is the development of FP-based biosensors, which change their fluorescence properties in response to a biological stimulus - such as enzymatic activity or changing ion concentrations. These molecules allow the dynamic observation of processes as they occur within the live cell, as has been done highly successfully for e.g. calcium (Ca2+) signaling. However, the combination of photophysically “smart” FPs and biosensors is an area of new and unexplored possibilities for super-resolution microscopy, optical highlighting and ratiometric imaging.The work presented here describes the development of novel and improved genetically encoded fluorescent reporters with expanded functionalities. Central to this work is the development of reversibly switchable genetically encoded calcium indicators (rsGECIs) by combining two concepts of FP technology: the photochromic behavior found in reversibly switchable FPs (RSFPs) and the FP-based calcium indicators used to visualize changing calcium concentrations.In the first chapter, I will present the research context and introduce the most important basic concepts and techniques regarding the work that follows. The next two chapters focus on my contributions to the improvement of well-known FPs for super-resolution fluorescence microscopy. In Chapter 2, I focus on the directed evolution of rsEGFP and describe the development and characterization of a family of improved-folding variants. These variants, the rsGreens, are applied in high-resolution imaging techniques such as pcSOFI and RESOLFT. In Chapter 3, I discuss how the non-phototransformable red fluorescent protein mCherry can be used to perform single-molecule localization microscopy via a chemically-induced blue-fluorescent state.Chapter 4 describes the first approach I used to create rsGECIs. Starting from two RSFPs with excellent photochromic behavior, rsGreen0.7 and Dronpa, I generated a series of structural variants of these FPs and integrated them into a GCaMP backbone. However, this strategy did not result in well-working probes.Chapter 5 discusses my efforts to develop new rsGECIs using a second strategy. Here, I started from the GCaMP family of calcium indicators and used rational mutagenesis to improve the photochromism in GCaMP. This resulted in the identification of a novel photochromic GCaMP mutant, which I used to determine the dynamically changing calcium concentrations in live cells. To achieve this, we also developed an advanced imaging scheme based on the calcium-dependent phototransformation of this new fluorescent indicator. This is followed by the final chapter, where I give the conclusions that could be drawn from this work and present future perspectives." "Visualization of biomolecular interactions and sensing in living systems with super-resolution fluorescence microscopy." "Johan Hofkens" "Molecular Imaging and Photonics" "Fluorescence microscopy is a powerful method to study living systems with high spatial and temporal resolution. However, the resolution of a conventional microscope is limited by diffraction, which precludes the direct visualization of many biological processes occurring at their small scale. During the last decade, several super-resolution fluorescence microscopy methods have been developed that break this diffraction limit and offer a new revolutionary view on structures with a size in the range of 100 nm and smaller. One of these techniques is pcSOFI, which distills sub-diffraction information out of a statistical analysis of blinking fluorophores.The necessary blinking for pcSOFI is most easily generated by using reversibly switchable fluorescent proteins (RSFPs), a class of photochromic derivatives of the green fluorescent protein (GFP), discovered in 1962. The special feature of these genetically encoded fluorophores is their capacity to reversibly switch between a fluorescent on-state and a non-fluorescent off-state, depending on the light with which they are irradiated. Continuous switching between the two states results in fluorescence blinking, suitable for pcSOFI analysis.Because of the key-role “photophysically smart labels”, such as RSFPs, play in super-resolution imaging, optimal performance of the methods is largely dependent on the quality of the used fluorophores. The development of optimized variants is thus a crucial step towards achieving the full potential of sub-diffraction microscopy.Within this dissertation, I outline the basics of how to create and characterize improved fluorescent proteins, and describe my efforts in developing RSFPs with beneficial properties for advanced imaging. In Chapter 2, I describe a series of mutants based on Dronpa and Dronpa2, which are a slow and a fast photoswitcher. By creating structural variation and optimizing the expression, I paved the way for the creation of refSOFI, a complementation approach that allows the visualization of protein-protein interactions with super-resolution. Other variants of Dronpa and Dronpa2 were shown to exhibit more fluorescence when expressed at 37°C and maintained efficient photoswitching characteristics.In Chapter 3, I introduce the rsGreens, which were developed using a strategy especially suited for optimizing “smart” fluorescent proteins. I provide an in depth characterization of a range of mutants, in terms of spectroscopic, photochromic and biological properties. The work on rsGreens is continued in Chapter 4, where I describe the structural analysis of rsGreen0.7 and the lessons learned about biological performance and photoswitching. This information is subsequently used for the structure-guided development of new rsGreen variants with significantly altered photoswitching characteristics.The final results chapter, Chapter 5, provides an extensive description of pcSOFI and includes an analysis of the method’s performance under different imaging conditions. I also present the first results obtained with multi-tau (mt) pcSOFI, which is a pcSOFI approach that can separate multiple spectrally similar fluorophores based on their blinking behavior.The development of a large number of new RSFPs, with beneficial biological and photochromic properties significantly increases the number of available “smart probes”. The characterization of the created RSFPs also contributes to a better understanding of the mechanisms behind all the different processes, which may benefit further developments. By aiding the development of refSOFI, by showing the potential of mt-pcSOFI and by performing the quality assessment of regular pcSOFI, I hope to have broadened the application area of pcSOFI and super-resolution microscopy in general." "Fluorescence microscopy tools for in situ catalyst characterization" "Maarten Roeffaers" "Centre for Surface Chemistry and Catalysis" "The goal of this PhD thesis is to apply fluorescence microscopy to investigate the interplay between local catalytic performance and catalyst porosity and to derive structure-activity and selectivity relationships for heterogeneous catalysts at the level of individual catalytic turnovers. To achieve this goal, I employed both diffraction-limited and super-resolution fluorescence microscopy with support from other techniques.A first objective was to study the effects of dealumination on the distribution of acid sites inside individual mordenite crystals and the impact hereof on the catalytic activity. Using super-resolution fluorescence microscopy, Raman microspectroscopy, and focused-ion-beam-assisted scanning electron microscopy I identified significant variations in catalytic properties inside individual dealuminated mordenites as well as strong variations between individual catalyst crystals. The origin of this suboptimal catalytic performance could be linked to variabilities that exist during commercial, large-scale dealumination.Secondly I studied the effect of solvents on the catalytic performance of acid H-ZSM-5. Using fluorescence microscopy with the acid-catalyzed furfuryl alcohol oligomerization reaction I discovered that the reaction preferentially occurs in a subset of the ZSM-5 pores. Using solvents of different polarity this pore selectivity could be altered. This result can be used to selectively perform catalytic reactions in either of the micropore subsystems.Later, this fluorescence based approach was extended to study the catalytic activity of metal-organic frameworks. For ZIF-8 I could prove that the reactivity is limited to the outer surface and bulk crystal defects. This inefficient use of the MOF material can be abated by the introduction of larger mesopores. In this project, I used oleic acid etching to increase the molecular penetration of the whole crystal volume.In conclusion, in this thesis I have applied fluorescence microscopy to resolve the structure-activity relationships in zeolites and metal-organic frameworks, and suggested strategies to optimize the catalytic activity. The results and the wealth of inferences therefrom demonstrate how fluorescence microscopy can enrich catalysis research as a characterization method. Such studies can be used to advance the field of catalyst design and development." "Multimodal fluorescence microscopy and nanoscopy platform." "Johan Hofkens" "Membrane Separations, Adsorption, Catalysis, and Spectroscopy for Sustainable Solutions (cMACS), Biochemistry, Molecular and Structural Biology, Molecular Imaging and Photonics" "Fluorescence microscopy and super-resolution fluorescence microscopy -nanoscopy- offers the possibility to study in a non-invasive way structures ultimately down to the nanometer scale. The ability to do so dynamically will prove to be an indispensable tool in analyzing the molecular mechanisms and organization of living as well as non-living systems. Technological advances in the microscopy field have now resulted in commercially available microscopes that allow fast imaging with light levels that do not perturb or damage the system at study which is e.g. crucial for living systems. At the same time, one needs to be able to put these super-resolution data into the context of the larger system, which requires a combination with and integration of confocal microscopy. The aimed for optical platform and the collaboration with the microscopy manufacturer Leica aims for future advances in combining maximal spatial resolution, dynamic imaging, context generation, development of quantitative imaging modes as well as tailor-made fluorescent probes. Such platform will be used to unravel the mode of operation of important research questions with societal relevance." "Advanced illumination techniques for high-contrast highresolution fluorescence and scattering microscopy" "Kristiaan Neyts" "Department of Electronics and information systems" "In light microscopy an object is illuminated with a lamp or a laser and the transmitted,reflected or fluorescent light is detected to get information on the object. In the project two approaches will be developed to study small particles and their interaction with living cells: Light Sheet Fluorescence Microscopy and Complex Beam Scattering Microscopy.In standard fluorescence microscopy the entire sample is illuminated and there is an important background arising from fluorescent material that is out of focus. In this project we will illuminate the sample with a sheet of light that coincides with the focal plane of the microscope, so that only that part of the sample that is in focus is illuminated. This can be realized with an optical fiber or by illuminating through the microscope objective lens onto an inclined mirror mounted on the sample holder. These solutions should be inexpensive and compatible with existing microscopes.For the study of small sub-resolution features in a sample we plan to scan specially designed focused laser beams near the object of interest and to detect the scattered light. The images of the scattered light are rich in information about the optical properties of the object of interest, such as size, shape and refractive index. In the project we will build a setup to scan different focused laser beams along an object, simulate scattering images for known objects and estimate the properties of unknown sub-resolution objects based on the scattering images." "High Resolution Structured Illumination Raster Scanning Fluorescence Microscopy" "Pol Van Dorpe" "Quantum Solid State Physics (QSP)" "Fluorescence microscopy has become an indispensable tool inbiology and medicine. It is routinely used in drug development, todiagnose health disorders and in DNA sequencing.Despite enormous progress in instrumentation and deployment, high-resolution microscopes remain expensive, bulky devices that requireskilled operators. As a consequence, they are found only inspecialized labs, limiting their accessibility.The next big push in microscopy with large societal impact will comefrom extremely compact and robust optical systems that will makehigh-resolution microscopy highly accessible.This push to miniaturization can be facilitated by photonic integratedcircuits—extremely compact chip-scale devices that are mass-produced using CMOS process technology.I will investigate a completely new approach towards high (diffractionlimited) resolution fluorescence microscopy that uses no lenses(lens-free) and is based on integrated visible photonics. All lens-based and state-of-the-art lens-free microscopy solutions use lightpropagation in 3D space to illuminate a sample. In this project’sapproach, the illumination is confined to the 2D world of a slab-waveguide. I will developed a method that allows to render acomplete high-resolution image within the constraints of the twodimensions of a photonic circuit integrated on a CMOS imager.This new technology will find many applications in life sciences andcompact, cost-effective DNA sequencing instruments."