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@PHDTHESIS{Sparrenberg:953448,
      author       = {Sparrenberg, Lorenz Tim},
      othercontributors = {Schwaneberg, Ulrich and Berlage, Thomas},
      title        = {{T}he analysis of fluorescence fluctuations by means of the
                      mean single-molecule rate (m{SMR})},
      school       = {RWTH Aachen University},
      type         = {Dissertation},
      address      = {Aachen},
      publisher    = {RWTH Aachen University},
      reportid     = {RWTH-2023-02644},
      pages        = {1 Online-Ressource : Illustrationen, Diagramme},
      year         = {2023},
      note         = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
                      University; Dissertation, RWTH Aachen University, 2023},
      abstract     = {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.},
      cin          = {162610 / 160000 / 122620 / 120000},
      ddc          = {570},
      cid          = {$I:(DE-82)162610_20140620$ / $I:(DE-82)160000_20140620$ /
                      $I:(DE-82)122620_20140620$ / $I:(DE-82)120000_20140620$},
      typ          = {PUB:(DE-HGF)11},
      doi          = {10.18154/RWTH-2023-02644},
      url          = {https://publications.rwth-aachen.de/record/953448},
}