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Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Max-Planck-Institut fu¨r Biophysikalische Chemie. (Nanobiophotonics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Show others and affiliations
2007 (English)In: Journal of Physical Chemistry A, ISSN 1089-5639, E-ISSN 1520-5215, Vol. 111, no 3, 429-440 p.Article in journal (Refereed) Published
Abstract [en]

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1−20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

Place, publisher, year, edition, pages
2007. Vol. 111, no 3, 429-440 p.
Keyword [en]
fluorescence correlation spectroscopy, photobleaching, antioxidants, mea, ascorbic acid, cot, npg
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-12331DOI: 10.1021/jp0646325ISI: 000243535700005Scopus ID: 2-s2.0-33846964579OAI: oai:DiVA.org:kth-12331DiVA: diva2:309766
Note
QC20100617Available from: 2010-04-12 Created: 2010-04-08 Last updated: 2017-12-12Bibliographically 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.
Series
Trita-FYS, ISSN 0280-316X ; 2010:20
Keyword
fluorescence correlation spectroscopy, triplet state, isomerisation, photobleaching, quenching, diffusion, total internal reflection, interface
National Category
Physical Chemistry
Identifiers
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)
Opponent
Supervisors
Note
QC 20100414Available from: 2010-04-14 Created: 2010-04-12 Last updated: 2011-01-20

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