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Time-Varying Excitation in Fluorescence Spectroscopy for Biological Applications
KTH, School of Engineering Sciences (SCI), Applied Physics.
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 [en]
fluorescence, spectroscopy, molecular kinetics
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-4399ISBN: 978-91-7178-713-2 (print)OAI: oai:DiVA.org:kth-4399DiVA: diva2:12132
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
List of papers
1. Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Open this publication in new window or tab >>Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Show others...
2007 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 9, 3330-3341 p.Article in journal (Refereed) Published
Abstract [en]

In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected

Keyword
Biomarkers; CCD cameras; Imaging systems; Molecular biology; Sensitivity analysis
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-7194 (URN)10.1021/ac0622680 (DOI)000246027100008 ()2-s2.0-34248153327 (Scopus ID)
Note

QC 20100805

Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2017-12-14Bibliographically approved
2. Modulated Fluorescence Correlation Spectroscopy with Complete Time Range Information
Open this publication in new window or tab >>Modulated Fluorescence Correlation Spectroscopy with Complete Time Range Information
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
Keyword
microscopy; kinetics; excitation; states
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-10237 (URN)10.1529/biophysj.107.113332 (DOI)000252243200024 ()2-s2.0-38849119190 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2017-12-13Bibliographically approved

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