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

Abstract

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