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Modulated Fluorescence Correlation Spectroscopy with Complete Time Range Information
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
2008 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 94, no 3, 977-985 p.Article in journal (Refereed) Published
Abstract [en]

Two methods to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times have been developed and experimentally verified. One method extracts distortion-free correlation data from measurements acquired with standard hardware correlators provided the fluorescence does not change systematically within the excitation pulses. This restriction does not apply to the second method, which, however, requires time-resolved acquisition of the fluorescence intensity. Modulation of the excitation in an FCS experiment is demonstrated to suppress triplet population buildup more efficiently than a corresponding reduction in continuous wave excitation intensity (shown for the dye rhodamine 6G in aqueous solution). Excitation modulation thus offers an additional means to optimize the FCS measurement conditions with respect to the photophysical properties of the dyes used. This possibility to suppress photoinduced states also provides a useful tool to distinguish additional processes occurring in the same time regime in the FCS measurements, as demonstrated here for the protonation kinetics of fluorescein at different pH. In general, the proposed concept opens for FCS measurements with a complete correlation timescale in a range of applications where a modulated excitation is either necessary or brings specific advantages.

Place, publisher, year, edition, pages
Bethesda, MD: the Biophysical Society , 2008. Vol. 94, no 3, 977-985 p.
Keyword [en]
microscopy; kinetics; excitation; states
National Category
Condensed Matter Physics
Identifiers
URN: urn:nbn:se:kth:diva-10237DOI: 10.1529/biophysj.107.113332ISI: 000252243200024Scopus ID: 2-s2.0-38849119190OAI: oai:DiVA.org:kth-10237DiVA: diva2:212165
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-11-15Bibliographically 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. Time-Varying Excitation in Fluorescence Spectroscopy for Biological Applications
Open this publication in new window or tab >>Time-Varying Excitation in Fluorescence Spectroscopy for Biological Applications
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The focus of this thesis is to explore and use the benefits of time-varying excitation in fluorescence spectroscopy for studies of biomolecular dynamics. Two new techniques taking advantage of modulated excitation are presented. Also described are the first efforts in a project where single molecule FRET and multi-parameter fluorescence detection are used for characterization of the conformational dynamics of the retinoid X receptor (RXR).

RXR is one of the most important proteins in the group of nuclear receptors. It is believed to be involved in many diseases and is hence most interesting as a potential drug target. Our study is at present at a very early stage and some sample issues are still to be resolved. However, single molecule measurements should give insights not attainable by previously applied ensemble methods and help explaining how RXR can regulate so many different processes.

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 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 imaging possible developments. The approach was experimentally verified by measurements of the triplet kinetics of rhodamine 6G (Rh6G) in aqueous solution and compared with fluorescence correlation spectroscopy (FCS). It should also be applicable to any other photoinduced transient states affecting the fluorescence intensity.

A strategy to combine 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 the measurement conditions with respect to the photophysical properties of the dyes used. Measurements were made on Rh6G to verify the method. To illustrate its usefulness, it was applied to measurements of protonation kinetics of fluorescein at different pH. FCS with modulated excitation will most probably prove very useful in many future studies involving multiple kinetic processes occurring in overlapping time ranges.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. vi, 46 p.
Series
Trita-FYS, ISSN 0280-316X ; 2007:38
Keyword
fluorescence, spectroscopy, molecular kinetics
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4399 (URN)978-91-7178-713-2 (ISBN)
Presentation
2007-06-14, FB54, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:15
Opponent
Supervisors
Note
QC 20101115Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2010-11-15Bibliographically approved

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