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Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
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 [en]
fluorescence, spectroscopy, microscopy, modulated excitation, intensity modulation, fluorescence correlation spectroscopy, transient states, molecular kinetics
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-10243ISBN: 978-91-7415-299-9 (print)OAI: oai:DiVA.org:kth-10243DiVA: diva2:213708
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
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
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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

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: 2014-09-24Bibliographically approved
2. Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy
Open this publication in new window or tab >>Transient State Imaging for Microenvironmental Monitoring by Laser Scanning Microscopy
2008 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 80, no 24, 9589-9596 p.Article in journal (Refereed) Published
Abstract [en]

Photoinduced transient dark states are exhibited by practically all common fluorophores. These relatively long-lived states are very sensitive to the local environment and thus highly attractive for microenvironmental imaging purposes. However, because of methodological constraints, their sensitivity has to date been very sparsely exploited. Here, a concept based on spatio-temporal modulation of the excitation intensity is presented that can image these states via their photodynamic fingerprints. With the use of a standard laser scanning microscope, it unites the outstanding environmental sensitivity of the transient state parameters with the high sensitivity of the fluorescence readout and is easily implemented. For demonstration, triplet state images of liposomes with different internal environments were generated. These images provide an example of bow local environmental differences can be resolved, which are not clearly distinguishable via other fluorescence parameters. Having minor instrumental and sample constraints the concept can be foreseen to provide several new, useful, and independent fluorescence-based parameters in biomolecular imaging.

Place, publisher, year, edition, pages
Washington, DC: American Chemical Society, 2008
Keyword
fluorescence correlation spectroscopy; absorption-spectroscopy; phosphorescence; dynamics
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-10236 (URN)10.1021/ac8018735 (DOI)000261728900027 ()2-s2.0-58149234147 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-12-07Bibliographically approved
3. 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: 2010-11-15Bibliographically approved
4. Modulation Filtering Enables Removal of Spikes in Fluorescence Correlation Spectroscopy Measurements without Affecting the Temporal Information
Open this publication in new window or tab >>Modulation Filtering Enables Removal of Spikes in Fluorescence Correlation Spectroscopy Measurements without Affecting the Temporal Information
2009 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 113, no 25, 8752-8757 p.Article in journal (Refereed) Published
Abstract [en]

The appearance of intensity spikes in measurements is a common problem in fluorescence correlation spectroscopy (FCS) studies of biological samples. In this work, we present a new method for generating artifact-free correlation curves from fluorescence traces that have undergone spike removal. This method preserves the temporal information throughout the measurement and properly represents the correlation between events separated by removed spikes. The method was validated using experimental data. The proposed algorithm is demonstrated herein to be generally applicable, but it is particularly powerful for cases where spikes occur frequently.

National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-10238 (URN)10.1021/jp902538b (DOI)000267205600045 ()2-s2.0-67649218418 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-12-06Bibliographically approved
5. Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
Open this publication in new window or tab >>Fluorescence cross-correlation spectroscopy of a pH-sensitive ratiometric dye for molecular proton exchange studies
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
Keyword
living cells; single molecules; excitation; microscopy; kinetics
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-10239 (URN)10.1039/b822494c (DOI)000266269200035 ()2-s2.0-65949124070 (Scopus ID)
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-12-06Bibliographically approved

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