Probing Molecular Stoichiometry by Photon Antibunching and Nanofluidics Assisted Imaging in Solution

by Hao Cheng
Doctoral thesis
Date of Examination:2017-05-18
Date of issue:2017-07-05
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Jörg Enderlein
Referee:Dr. Andreas Neef
Persistent Address: http://dx.doi.org/10.53846/goediss-6374

 

 

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Abstract

English

A mechanistic understanding of biological function requires a quantitative determination of macromolecular subunit architecture and interaction. Optical microscopy and spectroscopy provide a noninvasive method to characterize the stoichiometric ratios of molecular complexes. Though target-bound fluorescence labeling techniques can help to detect single molecules, counting molecules in a molecular complex remains challenging. In solution, diffusion limits the observation times of single molecules and, thus reduces the number of detectable photons. Current methods have limited resolving power or are constrained by a complex experimental configuration. Therefore, they are not able to precisely quantify the number of labeled fluorophores. In this dissertation, I first explore the ability of photon antibunching to probe molecular stoichiometry in solution. The underlying theoretical model is elucidated and subsequently applied to samples of different labeling stoichiometry. It enables determining the average number of emitters per molecular complex. In the second part of my thesis, to obtain the full distribution of species with a particular number of fluorescent labels, another method is developed. It is based on molecular brightness analysis using imaging-based photon counting histograms. This is assisted by a nanofluidic device that enables direct imaging of diffusing molecules with extended observation time. I performed a systematic study of the experimental conditions which guarantee an optimal performance of this method. The capability of correctly determining distributions of stoichiometries of molecular mixtures is verified by both simulation and measurements of small molecules. The nanofluidics system allows both single-molecule detection and manipulation under microscopic imaging, which is simple and implementation-friendly.
Keywords: single-molecule fluorescence; nanofluidics; brightness analysis; molecular stoichiometry; antibunching; stroboscopic imaging
 
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