PhD Thesis Defenses

PhD Thesis Defense: Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies


Fluorophore blinking dynamics are highly sensitive to the local environment and can be used as an additional readout parameter to increase the information gained from existing fluorescence techniques.The origin of these blinking patterns are photophysical transitions to and from a manifold of non-luminescent states. The long lifetime of these dark transient states, typically 103 to 106 times longer than the fluorescent state, gives them correspondingly more time to sense their environment. For this reason, fluorophore blinking dynamics are particularly sensitive to low frequency events, such as diffusion-mediated interactions between the fluorophore and dilute species.

Transient State (TRAST) monitoring has been developed to quantify fluorophore blinking dynamics in a simple and widely applicable manner. TRAST does not need to resolve individual blinking events, but instead monitors the average fluorescence intensity in response to a modulated excitation. By systematically varying the modulation parameters, the transient state kinetics of the sample are mapped out. Without the need for time-resolved detection, a regular camera can be used to image blinking dynamics with high spatial resolution.

This thesis presents TRAST characterizations of common autofluorescent compounds and demonstrates their ability to sense relevant biological parameters such as oxygen concentration and redox potential. In Papers I and II, the autofluorescent co-enzymes flavin and NAD(P)H were studied, and label-free imaging of local redox variations within cells was demonstrated. Perturbing the cells, through dilute additions of mitochondrial uncouplers, revealed a strong andlocalized response in the TRAST images. In Paper III we studied tryptophan autofluorescence and used it to detect conformational changes in an unlabeled spider silk protein.

Labeling with external fluorophores can add further specificity to the TRAST measurements. In Paper IV, TRAST was used to monitor diffusion-mediated interactions between lipids and receptors in a cell membrane, including the influence of receptor activation. In Paper V we tracked folding of RNA into G-quadruplexes in live cells, monitored via the isomerization properties of an attached cyanine dye