Connecting excited state lifetime and intensity correlations for quantitative analysis of chromophore interactions

Schröder, Tim (2022): Connecting excited state lifetime and intensity correlations for quantitative analysis of chromophore interactions. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Intensity correlation is a powerful tool to study intensity fluctuations on various time scales. Fluorescence correlation spectroscopy (FCS) is a popular representative to monitor spontaneous intensity fluctuations caused by the deviation of the system from thermal equilibrium at nanomolar concentrations. FCS is frequently used to determine local concentrations, diffusion coefficients and intermolecular interactions of fluorescently labeled biomolecules. However, determination of the forward and backward transition rate constants and thereby also the equilibrium constant is not possible when two intensity levels are involved e.g. in a Förster resonance energy transfer (FRET) experiment. The idea of combining the fluorescence lifetime information (microtime) with the intensity information (macrotime) lead to the development of fluorescence lifetime FCS (FLCS). However, FLCS requires prior knowledge of the fluorescence lifetime components and suffers in experiments from inaccurate bunching amplitudes. Therefore, it is the aim of this work to develop a model free connection between the microtime information and the macrotime information. To this end, shrinking-gate FCS (sg-FCS) is presented which enables the extraction of microscopic transition rate constants without prior knowledge by correlating photon subsets according to their arrival time after pulsed laser excitation. sg-FCS is demonstrated in simulations and in surface- and solution-experiments with a DNA based model system. Without prior knowledge, the equilibrium constant is recovered over two and a half orders of magnitude. Additionally, sg-FCS identifies dynamic bunching amplitudes in the intensity correlation as they come with a change in the fluorescence lifetime which is not the case for on-off switching processes. Beyond the analysis of photon bunching on long timescales, the degree of photon antibunching on short timescales is used as a metric for the number of emitters in a multichromophoric system. So far, the interpretation of antibunching has been hampered by exciton annihilation processes. On the one hand, singlet-singlet annihilation (SSA) increases the degree of photon antibunching. On the other hand, singlet-triplet annihilation (STA) results in photon bunching on longer timescales and the interpretation of photon antibunching in presence of photon bunching was not discussed in literature yet. Here, it is demonstrated in simulations and experiments with DNA origami-based model systems, how photon antibunching is affected by independent and collective chromophore blinking. Additionally, universal guidelines for correct interpretation of photon antibunching are identified. Thereby, the time dependence of the STA process is used to identify collective blinking chromophores by applying the sg-FCS analysis which in addition recovers the STA rate constant. In a similar approach, picosecond time resolved antibunching (psTRAB) also utilizes the excited state lifetime information which is then used to recover the true number of chromophores in a multichromophoric system which is subject to SSA. It is demonstrated in simulations and experiments, that psTRAB can recover the true number of emitters on a DNA origami structure besides efficient SSA. Additionally, the analysis reveals the dimensionality of exciton diffusion in mesoscopic H- and J-type conjugated polymer aggregates. At last, the potential for bright and small point light sources based on DNA origami is evaluated. State of the art dye loaded polymer beads suffer from inhomogeneous fluorescence properties and size. DNA origami provides stoichiometric and spatial control over the dye modifications and is a promising candidate to overcome the drawbacks of polymer beads. To obtain the highest labeling density on DNA origami structures the distance dependency of dye-dye interaction is systematically examined. At small distances, fluorescence lifetime and fluorescence intensity are quenched due to static and dynamic quenching which becomes less for larger distances until the dyes are permanently separated at ~ 3 nm distance. However, the dyes are not independent at this distances and resonant coupling like SSA as well as STA can affect the fluorescence intensity, photoblinking and photostability. All in all, the findings and algorithms described above are easy to apply in many laboratories around the world which are already using TCSPC and will contribute to the quantitative analysis of switching kinetics between intensity states by sg-FCS and the change of independent chromophore numbers over time psTRAB. At last, further consideration of weak and strong coupling effects between organic dyes in close proximity will pave the way to bright and unprecedented homogeneous DNA origami-based point light sources for biophysics experiments and super resolution microscopy.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: DNA origami, DNA nanotechnology, single molecule spectroscopy, interchromophoric interactions, photophysics, nanobeads, photon antibunching, fluorescence, microtime gating, intensity correlation, fluorescence correlation spectroscopy, FCS, singlet-singlet annihilation, microtime gating, photon statistics, quantum optics, time-gated fluorescence correlation spectroscopy, autocorrelation analysis, Förster resonance energy transfer, FRET
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. Mai 2022
• 1. Bericht­erstatter:in: Tinnefeld, Philip