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Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)ORCID iD: 0000-0003-3200-0374
2009 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 11, no 21, 4410-4418 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescence fluctuation analysis of individual pH-sensitive fluorophores has recently proven to be a useful approach for biomolecular proton exchange studies. In this work, dual-color fluorescence cross-correlation spectroscopy (FCCS) is demonstrated on a ratiometric pH-sensitive dye, for which both the excitation and emission spectra shift as a function of pH. In the FCCS measurements, the fluorescence signal from the predominant emission wavelength range of the protonated form of the dye is cross-correlated with that of the deprotonated form. Two lasers are used alternatingly to excite predominantly the protonated and the deprotonated form of the dye. The alternating excitation modulation scheme is combined with detection gating, and is based on a recently developed concept that allows extraction of correlation data for all correlation times regardless of the chosen modulation period. The scheme can thus be applied without concern for the time-scales of the molecular dynamic processes to be studied. By this combined discrimination based on both excitation and emission, spectral cross-talk is dramatically reduced and a very distinct and unambiguous anticorrelation can be recorded in the correlation curves as a consequence of the proton exchange. The strong discrimination power makes the approach applicable also to ratiometric dyes with less pronounced spectral shifts. It should also be useful in combination with ratiometric dyes sensitive to other ambient conditions and ions, such as the biologically very important calcium ion.

Place, publisher, year, edition, pages
Cambridge, UK: RSC Publishing , 2009. Vol. 11, no 21, 4410-4418 p.
Keyword [en]
living cells; single molecules; excitation; microscopy; kinetics
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-10239DOI: 10.1039/b822494cISI: 000266269200035Scopus ID: 2-s2.0-65949124070OAI: oai:DiVA.org:kth-10239DiVA: diva2:212186
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-12-06Bibliographically approved
In thesis
1. Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications
Open this publication in new window or tab >>Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine.

Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown.

A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems.

In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 81 p.
Series
Trita-FYS, ISSN 0280-316X ; 2009:13
Keyword
fluorescence, spectroscopy, microscopy, modulated excitation, intensity modulation, fluorescence correlation spectroscopy, transient states, molecular kinetics
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-10243 (URN)978-91-7415-299-9 (ISBN)
Public defence
2009-05-20, FB42, AlbaNova main building, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100805Available from: 2009-05-12 Created: 2009-04-21 Last updated: 2010-08-05Bibliographically approved
2. Monitoring Proton Exchange and Triplet States with Fluorescence
Open this publication in new window or tab >>Monitoring Proton Exchange and Triplet States with Fluorescence
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescent molecules commonly shift to transient dark states, induced bylight or triggered by chemical reactions. The transient dark states can beused as probes of the local environment surrounding the fluorescent molecules,and are therefore attractive for use in biomolecular applications. Thisthesis explores the use and development of novel fluorescence spectroscopictechniques for monitoring transient dark states.This work demonstrates that kinetic information regarding photoinduced transient dark states of fluorescent molecules can be obtained from the time-averaged fluorescence intensity of fluorescent molecules subject totemporally modulated illumination. Methods based on this approach havethe advantage that the light detectors can have a low time resolution, which allows for parallelization and screening of biomolecular interactions withhigh throughput. Transient state images are presented displaying local environmental differences such as those in oxygen concentration and quencher accessibility.Analysis of the fluorescence intensity fluctuations resulting from thetransitions to and from transient dark states can be used to obtain information regarding the transition rates and occupancy of the transient darkstates. Fluorescence fluctuation analysis was used to reveal rates of protonbinding and debinding to single fluorescent molecules located close to biological membranes and protein surfaces. The results from these studies show that the proton exchange rate increases dramatically when the fluorescent molecule is close to the membrane.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. vii, 79 p.
Series
Trita-FYS, ISSN 0280-316X ; 2009:14
Keyword
fluorescence correlation spectroscopy, proton transfer, cytochrome c oxidase, transient state imaging, modulated excitation
National Category
Atom and Molecular Physics and Optics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-10400 (URN)978-91-7415-304-0 (ISBN)
Public defence
2009-05-15, Sal FB42, AlbaNova, Roslagstullsbacken 21, Sstockholm, 09:00 (English)
Opponent
Supervisors
Note
QC 20100809Available from: 2009-05-13 Created: 2009-05-11 Last updated: 2011-01-24Bibliographically approved

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