The Mean Single Molecule Rate (mSMR) in the Analysis of Fluorescence Fluctuations: Measurements on DNA Mixtures of Defined Composition

Lorenz T Sparrenberg,Benjamin Greiner,Harald P Mathis

doi:10.1007/s10895-021-02803-3

Abstract

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.

Highlights

Fluorescence fluctuation spectroscopy (FFS) is an important tool to study biomolecules in solution
Subplot A shows the curves of the mean single molecule rate (mSMR) (T) for different single molecule brightness rates
In this paper we presented the evaluation of fluorescence fluctuation experiments based on Mandel’s Q parameter for increasing sampling times, making use of the concept of factorial cumulants for larger sampling times, as used in fluorescence intensity multi distribution analysis (FIMDA) and time integrated fluorescence cumulant analysis (TIFCA)

Summary

Introduction

Fluorescence fluctuation spectroscopy (FFS) is an important tool to study biomolecules in solution It is based on the statistical analysis of fluorescence intensity fluctuations due to the diffusion of fluorescent particles through an excitation volume. Background noise typically lowers the mSMR data and causes an underestimation ot the single molecule brightness rate. This effect is known in fluorescence correlation spectroscopy, where a decrease in the S/N ratio affects the average number of particles ⟨N⟩ in the detection volume. To compensate for this effect a correction term 2 is commonly used [24, 25]. We can adapt this term for the rate of the single molecule brightness as follows:

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Conclusion