The analysis of fluorescence fluctuations by means of the mean single-molecule rate (mSMR)

Dissertation, RWTH Aachen University, 2023; Aachen : RWTH Aachen University 1 Online-Ressource : Illustrationen, Diagramme (2023). = Dissertation, RWTH Aachen University, 2023
Fluorescence fluctuation spectroscopy (FFS) is an important tool for the analysis of biological systems at the single-molecule level. FFS methods can be roughly divided into two categories. Methods of the first category examine fluctuations in the time domain and include the well-known fluorescence correlation spectroscopy (FCS) and its variations. Methods of the other category analyze fluctuations in the amplitude domain and include the photon counting histogram and related methods. In this thesis, the mean single-molecule rate (mSMR) is introduced as a new method, which uses information from both the time and amplitude domain. The mSMR is based on Mandel’s Q parameter, which can be calculated from the first two cumulants of a fluorescence trace. The cumulants can be expressed for arbitrary sampling times of a fluorescence trace, which yields the Q parameter as a sampling time-dependent quantity. By normalizing the Q parameter to its corresponding sampling time, data curves are obtained which show great similarities to the autocorrelation curves in FCS analysis and enable a comparable interpretation of the data. The model definition based on cumulants allows direct correction of common detector artefacts such as afterpulsing or dead time. For evaluation, the mSMR is subjected to a series of systematic analyses. Firstly, it was applied to simulated fluorescence traces since the simulation enables precisely adjustable parameters. It was shown that the mSMR model accurately reproduces the input parameters of the simulation both in the absence and presence of noise and detector artefacts. Secondly, the mSMR was used to analyze fluorescence traces of the dye Alexa Fluor 488 recorded with a home-built confocal plate reader. Our reader automatically conducts FFS measurements in a microtiter plate, thus enabling easy and repeatable measurements with low hands-on time. A visual and statistical comparison between the mSMR and the established FCS showed that the mSMR provides generally comparable results to the FCS method. At low excitation powers and low concentrations, however, the mSMR provides more plausible results on short time scales. This is of particular importance for the analysis of photokinetic effects. Thirdly, to show the relevance of the mSMR for biological systems, measurements were performed on DNA mixtures of defined fragment length composition. Here, too, the mSMR retrieved precise results that are in line with theoretical expectations. Based on these findings, libraries for DNA sequencing were characterized and mass concentration, mean fragment length and molarity of the libraries were determined. In just one measurement, the mSMR could provide the same results as a commonly used multistep procedure consisting of fluorescence spectroscopy and capillary gel electrophoresis. The mSMR represents a meaningful extension of previous FFS methods. The findings of this work suggest that especially for measurements with few photon events, e.g., at low excitation powers and concentrations, the mSMR is a robust and reliable method. In combination with the correction of detector artefacts, the mSMR can resolve fluctuation events on very short time scales and permits high-precision analyses of fluorescence fluctuations. This provides new insights into the analysis of photokinetic effects.
Published by RWTH Aachen University, Aachen