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Recovery of Photoinduced Reversible Dark States Utilized for Molecular Diffusion Measurements
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2010 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 82, no 24, 9998-10005 p.Article in journal (Refereed) Published
Abstract [en]

For a spatially restricted excitation volume, the effective modulation of the excitation in time is influenced by the passage times of the molecules through the excitation volume. By applying an additional time-modulated excitation, the buildup of photoinduced reversible dark states in fluorescent molecules can be made to vary significantly with their passage times through the excitation volume. The variations in the dark state populations are reflected by the time-averaged fluorescence intensity, which thus can be used to characterize the mobilities of the molecules. The concept was experimentally verified by measuring the fluorescence response of freely diffusing cyanine fluorophores (Cy5), undergoingtrans-cis isomerization when subject to time-modulated excitation in a focused laser beam. From the fluorescence response, and by applying a simple photodynamic model, the transition times of the Cy5 molecules could be well reproduced when applying different laminar flow speeds through the detection volume. The presented approach puts no constraints on sample concentration, no requirements for high time resolution or sensitivity in the detection, nor requires a high fluorescence brightness of the characterized molecules. This can make the concept useful for a broad range of biomolecular mobility studies.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2010. Vol. 82, no 24, 9998-10005 p.
Keyword [en]
Fluorescence correlation spectroscopy, FCS, isomerisation, transient states, diffusion
National Category
Physical Chemistry Biophysics
Research subject
Biological Physics
URN: urn:nbn:se:kth:diva-12344DOI: 10.1021/ac1014047ISI: 000285215800006ScopusID: 2-s2.0-78650344728OAI: diva2:309816
EU, FP7, Seventh Framework Programme, 201 837Swedish Research Council, VR-NT 2009-3134Knut and Alice Wallenberg Foundation

QC 20100617 Uppdaterad från manuskript till artikel (20110110). Tidigare titel: Recovery of Photo-Induced Reversible Dark States Utilized for Molecular Diffusion Measurements

Available from: 2010-04-12 Created: 2010-04-08 Last updated: 2016-03-10Bibliographically approved
In thesis
1. Photo-induced dark states influorescence spectroscopy – investigations & applications
Open this publication in new window or tab >>Photo-induced dark states influorescence spectroscopy – investigations & applications
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. vii, 111 p.
Trita-FYS, ISSN 0280-316X ; 2010:20
fluorescence correlation spectroscopy, triplet state, isomerisation, photobleaching, quenching, diffusion, total internal reflection, interface
National Category
Physical Chemistry
urn:nbn:se:kth:diva-12372 (URN)978-91-628-8108-5 (ISBN)
Public defence
2010-04-29, FA31, Albanova University Center, Roslagstullsbacken 21, Stockholm, 13:00 (English)
QC 20100414Available from: 2010-04-14 Created: 2010-04-12 Last updated: 2011-01-20

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