Investigating the Impact of Single Molecule Fluorescence Dynamics on Photo Activated Localization Microscopy Experiments

When fluorophores are densely packed in a biological sample, localizing them one at a time is one of the paths to optical super-resolution. Photo Activated Localization Microscopy (PALM) is one of the methods of this burgeoning field that presents a large promise for addressing physical and biological questions requiring non-invasive, high-resolution optical imaging at the nanoscale. Since this work began when PALM was only two years old, the thesis contains a significant portion of research on the technique itself, and has therefore a marked methodological cut. The principal, and recurring, theme is the investigation of the single molecule fluorescence dynamics of the fluorophores used, that in the end determine to a large extent the ensemble imaging performance as well as the interpretation of the data. A few assumptions reported in the literature are questioned, and, beginning with a discussion of the properties of a bright fluorescent protein, mEos2, we propose an original approach based on the use of the temporal information besides the spatial one when treating PALM data. This allows both a more accurate quantification of the number of fluorophores activated in the sample as well as the correct identification of relevant biological structures, such as clusters, on the plasma membrane of cells. The second theme is the application of PALM to probe the functional arrangement of an important class of cell membrane proteins, through the study of the prototypical G protein-coupled receptor [beta]2-Adrenergic Receptor. First, the well studied biological properties of this family of signaling proteins are used to validate the potential of this approach. Then, PALM is used to gain new insight on the basal arrangement of [beta]2-AR in a relevant biological context using tools from spatial point pattern analysis. Provided that appropriate control experiments are performed, PALM is shown to have the potential to be included in the palette of the available techniques to study membrane protein organization. An important finding is the quantification of a cell-type specific clustering of this receptor in cardiomyocite-like cells. Finally, some technical issues relevant to dual color imaging, in particular concerning the axial stability of the microscope, are addressed as a third, perspective theme, together with a detailed first-time characterization of three representative fluorescent protein pairs currently available for dual color PALM imaging.