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Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics.ORCID iD: 0000-0002-5584-9170
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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

Place, publisher, year, edition, pages
2007. Vol. 79, no 9, 3330-3341 p.
Keyword [en]
Biomarkers; CCD cameras; Imaging systems; Molecular biology; Sensitivity analysis
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-7194DOI: 10.1021/ac0622680ISI: 000246027100008Scopus ID: 2-s2.0-34248153327OAI: oai:DiVA.org:kth-7194DiVA: diva2:12130
Note

QC 20100805

Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2017-12-14Bibliographically approved
In thesis
1. 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
2. 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
3. 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
4. Analysis of Fluorescence Flicker as a Tool to Monitor Proton Transport and Biomolecular Interactions
Open this publication in new window or tab >>Analysis of Fluorescence Flicker as a Tool to Monitor Proton Transport and Biomolecular Interactions
2006 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The overall focus of this thesis is on fluorescence flicker processes of fluorescent molecules, e.g. protonation-deprotonation or singlet-triplet electronic state transitions, intrinsic or generated by their interaction with their environment, monitored by fluorescence spectroscopy.

Understanding proton migration along membranes and membrane proteins in cells is essential for understanding energy metabolism. It has been seen that certain membrane-spanning proton-transporter proteins in the respiratory chain in the mitochondrial inner membrane take up protons faster than the rate limited by diffusion. To explain these observations it has been suggested that there is a proton-collecting antenna, consisting of negatively and protonatable residues on the surface of these proteins, which increases the rate of uptake. Using fluorescence correlation spectroscopy and artificial biological membranes the proton collecting antenna effect is verified, as well as the proton migration properties on these membranes at various surface buffer concentrations.

Fluorescence flicker due to singlet-triplet electronic state transitions in a fluorescent molecule is interesting because of the long transition time between the two states. This means that the molecule has a long time to interact with the local environment, and can therefore be used as a microenvironmental sensor. A novel method for monitoring photo-induced, transient, long-lived, non- or weakly fluorescent states, e.g. the triplet state, was developed. With this method, only the time averaged intensity is detected and used for determining the triplet state kinetics. This method has several advantages, in particular it lends itself well for parallelization, over traditional methods including fluorescence correlation spectroscopy.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. iv, 30 p.
Series
Trita-FYS, ISSN 0280-316X ; 2006:75
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4216 (URN)978-91-7178-546-6 (ISBN)
Presentation
2006-12-20, FA31, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00
Opponent
Supervisors
Note
QC 20101126Available from: 2006-12-07 Created: 2006-12-07 Last updated: 2010-11-26Bibliographically approved

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Publisher's full textScopushttp://pubs.acs.org/doi/abs/10.1021/ac0622680

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Blom, Hans

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