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- All Subjects: HMM
- All Subjects: Bayesian nonparametric
- All Subjects: Fluorescence microscopy--Mathematical models.
- Creators: Presse, Steve
- Creators: Pollat, Scott
Single molecule FRET experiments are important for studying processes that happen on the molecular scale. By using pulsed illumination and collecting single photons, it is possible to use information gained from the fluorescence lifetime of the chromophores in the FRET pair to gain more accurate estimates of the underlying FRET rate which is used to determine information about the distance between the chromophores of the FRET pair. In this paper, we outline a method that utilizes Bayesian inference to learn parameter values for a model informed by the physics of a immobilized single-molecule FRET experiment. This method is unique in that it combines a rigorous look at the photophysics of the FRET pair and a nonparametric treatment of the molecular conformational statespace, allowing the method to learn not just relevant photophysical rates (such as relaxation rates and FRET rates), but also the number of molecular conformational states.
Several experimental measurements can probe diffusion coefficients at the single-molecule and bulk level. The target of this thesis is on single-molecule methods, which can assess diffusion coefficients at the individual molecular level. For instance, super resolution methods like stochastic optical reconstruction microscopy (STORM) and photo activated localization microscopy (PALM), have a high spatial resolution with the cost of lower temporal resolution. Also, there is a different group of methods, such as MINFLUX, multi-detector tracking, which can track a single molecule with high spatio-temporal resolution. The problem with these methods is that they are only applicable to very diluted samples since they need to ensure existence of a single molecule in the region of interest (ROI).
In this thesis, the goal is to have the best of both worlds by achieving high spatio-temporal resolutions without being limited to a few molecules. To do so, one needs to refocus on fluorescence correlation spectroscopy (FCS) as a method that applies to both in vivo and in vitro systems with a high temporal resolution and relies on multiple molecules traversing a confocal volume for an extended period of time. The difficulty here is that the interpretation of the signal leads to different estimates for the kinetic parameters such as diffusion coefficients based on a different number of molecules we consider in the model. It is for this reason that the focus of this thesis is now on using Bayesian nonparametrics (BNPs) as a way to solve this model selection problem and extract kinetic parameters such as diffusion coefficients at the single-molecule level from a few photons, and thus with the highest temporal resolution as possible.