Restriction in the outer membrane diffusion over the dividing septum of Escherichia coli cells measured by dual-color FRAP

In this thesis, fluorescence spectroscopy was utilized to probe protein and lipid dynamics and interactions in their native, or close to native, environments. Thereby insight could be gained into the fundamentals of bacterial cell division and the innateimmune system. A particular focus has been devoted to fluorescence fluctuations. They arise when a lower number of fluorescent molecules undergo Brownian motion through a confocal detection volume. With sensitive detectors, appropriate optics and efficient data acquisition these fluctuations can be observed and correlated. This is the heart in fluorescence correlation spectroscopy (FCS), conceived in the 1970’s. FCS has the power to quantify absolute concentrations, diffusion coefficients and to some extent binding events, and is suitable for measurements on a range of samples, including living cells. However, FCS is not limited to solely diffusional processes. The sensitivity and time resolution allows also electron transitions within the fluorescent molecules to be probed. Of particular interest are spin transitions to and from the dark triplet state. This state is long-lived and sensitive, making it an effective sensor of the surrounding environment. We found that the triplet state could also be used to probe low frequency interactions in membranes down to a single molecule level and a theoretical model was developed that supported the observed interactions. FCS can be extended to fluorescence cross-correlation (FCCS), to handle also a second type of fluorescent molecule, fluorescing with a different colour, and whose signal is cross-correlated with the signal from the first type of fluorescent molecules. The aim was to improve and apply FCCS as a screening tool to probe proteinprotein interactions. By utilizing a methodology based on fluorescently labelled antibodies, we were able to calibrate our FCCS system and provide quantitative data on a particular receptor interaction occurring in natural killer cells. The methodology was found to increase the possibility to quantitatively analyse protein-protein interactions in the membrane of living cells.