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Determination of molecular stoichiometry without reference samples by analysing fluorescence blinking with and without excitation synchronization
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

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.

Keyword [en]
FCS; transient state; stoichiometry; TRAST; photo-switching
National Category
Natural Sciences Biophysics
Research subject
Biological Physics
URN: urn:nbn:se:kth:diva-144061OAI: diva2:710753
VR-NT, 2012-3045
Swedish Research Council, VR-NT, 2012-3045Knut and Alice Wallenberg Foundation, 2011.0218

QS 2014

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2016-03-10Bibliographically approved
In thesis
1. Fluorescence-based methods to probe long-lived transient states for biomolecular studies
Open this publication in new window or tab >>Fluorescence-based methods to probe long-lived transient states for biomolecular studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging are wide-spread tools in life science. Themain read-out parameters are still fluorescence intensity and wavelength, but given thebenefits of multi-parameter characterization there are also good reasons to consideradditional fluorescence-based read-out parameters. A major focus of this thesis is toextend the use of transient, non-fluorescent states as additional parameters forbiomolecular studies. To-date, such states (including mainly triplet states, isomerizedstates and photo-ionized states) have been exploited to a very limited extent for thispurpose. Their use has been limited because they show very weak, or no luminescence atall, and absorption measurements require relatively complex instrumentation which aretypically not applicable for studies under biologically relevant conditions. Moreover, thelong lifetime of these transient states make any readout signal very sensitive to changes inthe micro-environment, e.g. presence of small amounts of quenchers, like oxygen. Thosetransient states can be accessed by fluorescence correlation spectroscopy (FCS) and thenewly developed transient state (TRAST) monitoring technique. In this thesis, FCS andTRAST have been applied to demonstrate the use of transient state monitoring forbiomolecular studies.

In Paper I, we demonstrated that due to the low brightness requirements ofTRAST, also autofluorescent molecules like tryptophan can be studied, making externallabeling of molecules redundant. The photo-physical transient states of tryptophan andtryptophan-containing proteins could be analyzed and were found to provide informationabout protein conformational states and about the influence of pH and buffers on singletryptophan molecules. In Paper II investigations of the transient states of theoligothiophene p-FTAA with FCS as well as with dynamic light scattering andspectrofluorimetry revealed a pH dependent aggregation behavior and a very efficientfluorescence quenching by oxygen could be identified and analyzed. In Paper III, FCSand TRAST were used to monitor the isomerization kinetics of Merocyanine 540incorporated in lipid membranes. Because isomerization of cyanine dyes strongly dependson the viscosity of the local environment, the isomerization kinetics could be used tocharacterize membrane fluidity in artificial lipid vesicles and in cellular membranes. InPaper IV, a new approach was developed, based on a combination of TRAST and FCS todetermine the stoichiometry of a fluorescently labeled sample. Finally, in Paper V, FCSand TRAST were employed to demonstrate that triplet states of fluorophores can provide auseful readout for Förster Resonance Energy Transfer (FRET) reflecting intra- orivintermolecular distances between two fluorophores. The sensitivity of the triplet statemade it possible to monitor distances larger than 10 nm, which is often stated as the upperlimit of FRET interactions.

Taken together, the studies presented in this thesis show that there is a wealth ofinformation that can be revealed by studying long-lived transient states. Both FCS andTRAST combine a sensitive readout via the fluorescence signal with the sensitivity of thelong-lived transient states monitored via the fluorescence changes. It can therefore bepredicted that these approaches will find additional applications in the future.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xii, 49 p.
TRITA-FYS, ISSN 0280-316X ; 2014:14
National Category
Natural Sciences
Research subject
urn:nbn:se:kth:diva-144063 (URN)978-91-7595-104-1 (ISBN)
Public defence
2014-04-29, FA31, Roslagstullsbacken 21, Stockholm, 09:00 (English)

QC 20140408

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2014-04-08Bibliographically approved

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The manuscript is published 2015 in Methods and Applications in Fluorescence

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