The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition.
Cumulant analysis
Diffusion
Fluorescence fluctuation spectroscopy
Nucleic acids
Single molecule brightness
Journal
Journal of fluorescence
ISSN: 1573-4994
Titre abrégé: J Fluoresc
Pays: Netherlands
ID NLM: 9201341
Informations de publication
Date de publication:
Nov 2021
Nov 2021
Historique:
received:
22
03
2021
accepted:
11
08
2021
pubmed:
17
9
2021
medline:
4
2
2022
entrez:
16
9
2021
Statut:
ppublish
Résumé
We present a method for the evaluation of fluorescence fluctuations on the basis of Mandel's Q parameter, using sampling time-dependent factorial cumulants. By relating the Q parameter to the sampling time, we obtain the mean single molecule rate (mSMR), an easy to interpret expression that provides both brightness and diffusion information. The model is suitable for the widely used confocal setups with single photon excitation and a single detection channel. We present a way to correct the mSMR for afterpulsing, dead time and background noise. To account for photokinetic effects at short sampling times, we expand the model by a simple on/off isomerization term, which is similar to the well-known triplet model. The functionality of the mSMR is shown using Monte Carlo simulations. The correction mechanisms and the experimental applicability of the model are then demonstrated by DNA measurements of defined composition. By systematically analyzing DNA mixtures, we can show that at large sampling times, the mSMR correctly describes the single molecule brightness rates and the diffusive properties of DNA molecules. At short sampling times, the photokinetic effects of isomerization are accurately described by the mSMR model. Since additionally the mSMR can easily be corrected for measurement artefacts such as detector dead time, afterpulsing and background noise, this is a valuable advantage over the standard method of fluorescence correlation spectroscopy.
Identifiants
pubmed: 34529200
doi: 10.1007/s10895-021-02803-3
pii: 10.1007/s10895-021-02803-3
pmc: PMC8547212
doi:
Substances chimiques
Fluorescent Dyes
0
DNA
9007-49-2
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1883-1894Informations de copyright
© 2021. The Author(s).
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