Single-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in... Show moreSingle-Molecule Microscopy (SMM) techniques constitute a group of powerful imaging tools that enable researchers to study the dynamic behavior of individual molecules.In the research described in this doctoral thesis, SMM techniques have been developed to image individual proteins inside cells of a living zebrafish embryo model and to study patterns of their mobility.The results of the mobility pattern analyses offer new insights into the dynamics of single molecules diffusing inside cells within the context of an intact vertebrate organism. Show less
In this thesis we investigated the ability of two-photon multifocal microscopy for single-molecule microscopy in live cells and organisms. Two-photon excitation combined with multifocal scanning... Show moreIn this thesis we investigated the ability of two-photon multifocal microscopy for single-molecule microscopy in live cells and organisms. Two-photon excitation combined with multifocal scanning has the potential to achieve, high (temporal) resolution imaging at a low background. Splitting the laser beam into multiple beamlets reduced laser power to all but eliminate photobleaching. The low background combined with the fast scan speeds and absence of photobleaching allowed us to measure single-particles and single-molecules in live zebrafish embryo's for long time periods. To the best of our knowledge, it is the first time that single-molecule molecules have been observed in a wide-field two-photon microscope. Show less
Chemotaxis, the process in which cells detect a concentration gradient of a specific substance, interpret that information, and subsequently initiate movement towards the source is an essential... Show moreChemotaxis, the process in which cells detect a concentration gradient of a specific substance, interpret that information, and subsequently initiate movement towards the source is an essential part of many biological phenomena. It___s central to the processes in wound healing, in immune defense and in the formation of a viable embryo. In this thesis I used the well characterized social amoeba Dictyostelium discoideum to investigate, in depth, the dynamics that govern the first steps in the detection of a chemical gradient. D. discoideum detects cyclic adenosine mono-phosphate (cAMP) by a special receptor protein, cAMP receptor 1 (cAR1). Inside the cell this receptor activates a G protein which subsequently initiates a complex signaling cascade. Using fluorescence single-molecule microscopy I investigated the movements of both cAR1 and its associated G protein. During chemotaxis both proteins show striking differences in mobility between the leading and trailing edge of the cell. Those differences are presumably key to our understanding of gradient sensing by cells that have been ignored in models so far. Show less
The behavior of single G-protein coupled receptor molecules were studied with single-molecule microscopy in the plasmamembrane during Dictyostelium discoideum chemotaxis. The mobility of the... Show moreThe behavior of single G-protein coupled receptor molecules were studied with single-molecule microscopy in the plasmamembrane during Dictyostelium discoideum chemotaxis. The mobility of the receptor was different in the anterior and posterior regions of living cells migrating towards the source of chemoattractant. This difference of mobility can account for an amplification of the extracellular signal, necessary for chemotaxis. It was also found that the receptor molecules are internalized. Show less
