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

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