PhD Thesis Defenses

Concepts and biomedical applications of excitation-modulated transient state monitoring of fluorescence emitters


Fluorescence methods have developed very strongly in the last decade, allowing single- molecule detection sensitivity, high specificity, high time- and spatial resolution, as well as high readout speeds. Together, this makes fluorescencea very central readout modality for biomolecular and cellular studies. The photophysics of the fluorescence emitters, fluorophores, used is of central importance or the performance. Here, the photostability and brightness ofthe fluorophores, but also their blinking properties set the limits of the performance. Fluorophore blinking, arising from photo-induced, non-fluorescent transient states of fluorophores, can however also be taken advantage of cellular and biomolecular studies. Blinking is a prerequisite for essentially allso-called fluorescence-based super-resolution techniques. Moreover, blinkingis often sensitive to micro-environmental parameters such as pH, oxygen concentrations,redox conditions and viscosity. This follows from the fact that the underlying, non-luminescent, dark transient states typically have 103 to 106 longer lifetimes than the fluorescent excited states of the fluorophores, thereby giving fluorescent molecules in the dark states more time to interactwith their surrounding in biological environment. It can thus be utilized as an alternative readout parameter to provide useful information on molecules and cells and their environments, beyond what can be monitored by traditional fluorescence methods. This thesis takes as one starting point the transient state (TRAST) spectroscopy technique, designed to monitor such long-lived, dark transient states, including triplet, photo-oxidation, photo-reduction and photo-isomerized states of fluorophores, by measuring how the time-averaged fluorescence signal detected in the sample is changed upon systematically varying the excitation modulation.

The major focus of the present thesis work is to further extent the use of long-lived dark transient states of fluorescence emitters in solution, lipid membranes and live cells. For this different TRAST modalities were adapted and developed, and then demonstrated as useful characterization methods. First, we showed how the relaxed brightness requirements of TRAST made it possible to characterize the photo-physical properties of the high triplet yield carboxy-fluorescein dye and its brominated derivatives (paper I). By widefield TRAST measurements, we demonstrated its capability to sense heavy atom effect of bromine and iodide atoms, and how they affected the triplet and long-lived photo-oxidation states and their transitions rates. Next, we developed and demonstrated a concept based on TRAST method and its ability to distinguish fluorophores with different blinking properties as a way to perform fluorescence-based barcoding and multiplexing. This concept, demonstrated by exploiting the by TRAST well distinguishable photophysical transitions of two fluorescent dyes, which emit in the same spectral range, was demonstrated in paper II. In the same work, we also developed a TRAST modality for microfluidic measurements of molecules and lipid vesicles, on which the bar-coding concept could be demonstrated on-the-fly, as the molecules and vesicles passed through the microfluidic channel. Furthermore, with TRAST implemented in camera-based wide-field microscopy, multicolor barcoded images of cells with high spatial resolution could be further investigated for the first time due to the specific blinking dynamics of these labels. The last two papers of this thesis describe further extensions of the TRAST concept, and the monitoring of fluorescence blinking to live cell studies. In paper III, TRAST in a widefield microscopy setting was employed in combination with FCS to study the folding of dye-labelled RNA strands into G-quadruplex structures in solution and live cells using photo-isomerization kinetics of cyanine dye as a readout parameter. Here, we took advantage of the high sensitivity of cyanine dye photoisomerization, to viscosity and steric constraints,and the resulting blinking of the cyanines, to monitor conformation changes of RNAs in live cells. Finally, in paper IV, we demonstrated how it by TRAST imaging, taking advantage of the photo-induced dark states of a mitochondrial localization fluorophore (n-Nonyl Acridine Orange, NAO), is possible to give this localization probe environmental sensing properties as well.

To sum up, the experimental findings and papers included in this thesis show that fluorescence blinking represent a rich source of information for biomolecular and cellular studies. By the TRAST technique, and the variants further adapted and developed in this work, it is shown that it possible tocapture this rich source of complementary information in a broad range of samples. The work in this thesis suggest that further combination of classical fluorescence readouts and a continued development of different TRAST modalities will open yet new windows and provide insights into molecular interaction studies in biological research.