Determination of molecular stoichiometry without reference samples by analyzing fluorescence blinking with and without excitation synchronization

Abstract

Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study.The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.

Fluorescence Bar-Coding and Flowmetry Based on Dark State Transitions in Fluorescence Emitters

“…While CF640R-SUVs yielded similar fitted k isc and k T rates as for free CF640R, the fitted isomerization rates determined for Cy5-labeled SUVs were lower than for free Cy5 ( k iso = 6.2 μs –1 and σ biso = 0.042 × 10 –16 cm 2 ) but showed similar dark-state amplitudes, A . A did not increase with lower fractions of Cy5-labeled lipids in the SUV, indicating that the probability that any of the SUVs contained more than one fluorophore was negligible . The fitted rate parameter values from the FCS measurements of Cy5-SUVs and CF640R-SUVs, as well as for free Cy5 and CF640R, are summarized in Supplementary section S7.…”

“…A did not increase with lower fractions of Cy5-labeled lipids in the SUV, indicating that the probability that any of the SUVs contained more than one fluorophore was negligible. 30 The fitted rate parameter values from the FCS measurements of Cy5-SUVs and CF640R-SUVs, as well as for free Cy5 and CF640R, are summarized in Supplementary section S7 . For CF640R-SUVs and Cy5-SUVs, Q CF – SUV and Q Cy 5– SUV were found to be the same.…”

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

“…Figure 4 b illustrates a concept to determine molecular stoichiometries, by comparing the fluorescence fluctuations under continuous excitation using FCS, when all the fluorophores on a diffusing unit are blinking independently of each other, with those occurring under square-pulsed excitation using TRAST spectroscopy, when all fluorophores are blinking in a synchronized manner. Thereby, the number of fluorophores per molecule can be determined (Hevekerl and Widengren 2015 ). In the recorded FCS curves, the fast relaxation amplitude, B , of a transient state, is lowered with the number of independently blinking emitters, N , on a diffusing unit by B / N .…”