Qualification exams

Qualification exam Sune Christensen and Andreas H. Kunding.

10:00-11:00:
Andreas H. Kunding: Quantitative imaging of surface-interacting vesicles

With the invention of new and powerful fluorescence imaging setups, such as stimulated emission depletion, 4PI as well as the more established platforms of confocal laser scanning and total internal reflection fluorescence, the discipline of microscopy is now getting more and more quantitative. Intensities at different wavelengths constitute the raw data from which all fluorescence micrographs are built. Intensity values are a signature of the imaged object and can provide information of nanoscopic features even below the diffraction limited optical resolution. In this presentation confocal laser scanning microscopy (CLSM) and total internal fluorescence (TIRF) microscopy is applied to quantify populations of surface-immobilized vesicles.
The most common vesicle preparation technique; extrusion of freeze/thawed samples is characterized by CLSM with the intention of investigating the effects of filter pore dimension and rehydration method. Applying the results from CLSM transiently surface-bound vesicles are studied with TIRF microscopy to determine size dependent modes of binding. 

11:00-12:00:
Sune Christensen: Single vesicle assaying of membrane fusion

The controlled fusion of phospholipid membranes is a process central to the development of higher order life forms. A highly conserved family of proteins called SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) has been found to be involved in nearly all cellular fusion pathways. Ensemble based in vitro assays monitoring the fusion of vesicles reconstituted with v- and t-SNAREs have been widely adopted in the study of biological membrane fusion. However, an inherent drawback of this approach remains the inability to resolve each step of the fusion reaction, i.e. docking, priming, hemifusion and full fusion. Here I report on the design of an experimental platform that allows the tracking of single vesicle fusion events in real time. The invented assay was applied in a study of (i) the fusion of oppositely charged vesicles and (ii) fusion mediated by neuronal SNAREs together with the calcium sensor synaptotagmin-1.  

Supervisor: Dimitrios Stamou, co-supervisor: Thomas Bjørnholm 

Censor: Assoc Prof. Luis Bagatolli, Univerisity of Southern Denmark.