Improving FRAP and SPT for mobility and interaction measurements of molecules and nanoparticles in biomaterials

An increasing amount of pharmaceutical technologies are being developed in which nanoparticles play a crucial role. The rational development of these technologies requires detailed knowledge of the mobility and interaction of the nanoparticles inside complex biomaterials. The aim of this PhD thesis is to improve fluorescence microscopy based methods that allow to extract this information from time sequences of images. In particular, the fluorescence microscopy techniques Fluorescence Recovery After Photobleaching (FRAP) and Single Particle Tracking (SPT) are considered. FRAP modelling is revisited in order to incorporate the effect of the microscope's scanning laser beam on the shape of the photobleached region. The new model should lead to more straightforward an accurate FRAP measurements. SPT is the main focus of the PhD thesis, starting with an investigation of how motion during image acquisition affects the experimental uncertainty with which the nanoparticle positions are determined. This knowledge is used to develop a method that is able to identify interactions between nanoparticles in high detail, by scanning their trajectories for correlated positions. The method is proven to be useful in the context of drug delivery, where it was used to study the intracellular trafficking of polymeric gene complexes. Besides SPT data analysis, it is also explored how light sheet illumination, which allows to strongly reduce the out of focus fluorescence that degrades the contrast in SPT experiments, can be generated by a planar waveguide that is incorporated on a disposable chip. The potential as platform for diagnostic measurements was demonstrated by using the chip to perform SPT size and concentration measurements of cell-derived membrane vesicles. The results of this PhD thesis are expected to contribute to the effort of making accurate SPT and FRAP measurements of nanoparticle properties in biomaterials more accessible to the pharmaceutical research community

FRAP in pharmaceutical research: practical guidelines and applications in drug delivery

Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscopy technique that has attracted a lot of interest in pharmaceutical research during the last decades. The main purpose of FRAP is to measure diffusion on a micrometer scale in a non-invasive and highly specific way, making it capable of measurements in complicated biomaterials, even in vivo. This has proven to be very useful in the investigation of drug diffusion inside different tissues of the body and in materials for controlled drug delivery. FRAP has even found applications for the improvement of several medical therapies and in the field of diagnostics. In this review, an overview is given of the different applications of FRAP in pharmaceutical research, together with essential guidelines on how to perform and analyse FRAP experiments

Photopolymerized thermosensitive poly(HPMAlactate)-PEG-based hydrogels : effect of network design on mechanical properties, degradation, and release behavior

Photopolymerized thermosensitive A-B-A triblock copolymer hydrogels composed of poly(N-(2-hydroxypropyl)-methacrylamide lactate) A-blocks, partly derivatizal with methacrylate groups to different extents (10, 20, and 30%) and hydrophilic poly(ethylene glycol) B-blocks of different molecular weights (4, 10, and 20 kDa) were synthesized. The aim of the present study was to correlate the polymer architecture with the hydrogel properties, particularly rheological, swelling, degradation properties and release behavior. It was found that an increasing methacrylation extent and a decreasing PEG molecular weight resulted in increasing gel strength and cross-link density, which tailored the degradation profiles from 25 to more than 300 days. Polymers having small PEG blocks showed a remarkable phase separation into polymer- and water-rich domains, as demonstrated by confocal microscopy. Depending on the hydrophobic domain density, the loaded protein resides in the hydrophilic pores or is partitioned into hydrophilic and hydrophobic domains, and its release from these compartments is tailored by the extent of methacrylation and by PEG length, respectively. As the mechanical properties, degradation, and release profiles can be fully controlled by polymer design and concentration, these hydrogels are suitable for controlled protein release

Successful optimization of reconstruction parameters in structured illumination microscopy

The impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) on image artifacts is carefully analyzed. These parameters comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted. © 2018 The Author

Complementarity of PALM and SOFI for super-resolution live cell imaging of focal adhesions

Live cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenging task for super-resolution microscopy. We have addressed this important issue by combining photo-activated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed cell focal adhesion images, we investigated the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework was used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualized the dynamics of focal adhesions, and revealed local mean velocities around 190 nm per minute. The complementarity of PALM and SOFI was assessed in detail with a methodology that integrates a quantitative resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as the fluorophore density and the photo-activation and photo-switching rates

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells

Investigating Focal Adhesion Substructures by Localization Microscopy

Cells rely on focal adhesions (FAs) to carry out a variety of important tasks, including motion, environmental sensing, and adhesion to the extracellular matrix. Although attaining a fundamental characterization of FAs is a compelling goal, their extensive complexity and small size, which can be below the diffraction limit, have hindered a full understanding. In this study we have used single-molecule localization microscopy (SMLM) to investigate integrin β3 and paxillin in rat embryonic fibroblasts growing on two different extracellular matrix-representing substrates (i.e., fibronectin-coated substrates and specifically biofunctionalized nanopatterned substrates). To quantify the substructure of FAs, we developed a clustering method based on expectation maximization of a Gaussian mixture that accounts for localization uncertainty and background. Analysis of our SMLM data indicates that the structures within FAs, characterized as a Gaussian mixture, typically have areas between 0.01 and 1 μm2 , contain 10–100 localizations, and can exhibit substantial eccentricity. Our approach based on SMLM opens new avenues for studying structural and functional biology of molecular assemblies that display substantial varieties in size, shape, and density