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Fluorescence-based methods to probe long-lived transient states for biomolecular studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging are wide-spread tools in life science. Themain read-out parameters are still fluorescence intensity and wavelength, but given thebenefits of multi-parameter characterization there are also good reasons to consideradditional fluorescence-based read-out parameters. A major focus of this thesis is toextend the use of transient, non-fluorescent states as additional parameters forbiomolecular studies. To-date, such states (including mainly triplet states, isomerizedstates and photo-ionized states) have been exploited to a very limited extent for thispurpose. Their use has been limited because they show very weak, or no luminescence atall, and absorption measurements require relatively complex instrumentation which aretypically not applicable for studies under biologically relevant conditions. Moreover, thelong lifetime of these transient states make any readout signal very sensitive to changes inthe micro-environment, e.g. presence of small amounts of quenchers, like oxygen. Thosetransient states can be accessed by fluorescence correlation spectroscopy (FCS) and thenewly developed transient state (TRAST) monitoring technique. In this thesis, FCS andTRAST have been applied to demonstrate the use of transient state monitoring forbiomolecular studies.

In Paper I, we demonstrated that due to the low brightness requirements ofTRAST, also autofluorescent molecules like tryptophan can be studied, making externallabeling of molecules redundant. The photo-physical transient states of tryptophan andtryptophan-containing proteins could be analyzed and were found to provide informationabout protein conformational states and about the influence of pH and buffers on singletryptophan molecules. In Paper II investigations of the transient states of theoligothiophene p-FTAA with FCS as well as with dynamic light scattering andspectrofluorimetry revealed a pH dependent aggregation behavior and a very efficientfluorescence quenching by oxygen could be identified and analyzed. In Paper III, FCSand TRAST were used to monitor the isomerization kinetics of Merocyanine 540incorporated in lipid membranes. Because isomerization of cyanine dyes strongly dependson the viscosity of the local environment, the isomerization kinetics could be used tocharacterize membrane fluidity in artificial lipid vesicles and in cellular membranes. InPaper IV, a new approach was developed, based on a combination of TRAST and FCS todetermine the stoichiometry of a fluorescently labeled sample. Finally, in Paper V, FCSand TRAST were employed to demonstrate that triplet states of fluorophores can provide auseful readout for Förster Resonance Energy Transfer (FRET) reflecting intra- orivintermolecular distances between two fluorophores. The sensitivity of the triplet statemade it possible to monitor distances larger than 10 nm, which is often stated as the upperlimit of FRET interactions.

Taken together, the studies presented in this thesis show that there is a wealth ofinformation that can be revealed by studying long-lived transient states. Both FCS andTRAST combine a sensitive readout via the fluorescence signal with the sensitivity of thelong-lived transient states monitored via the fluorescence changes. It can therefore bepredicted that these approaches will find additional applications in the future.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , xii, 49 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:14
National Category
Natural Sciences
Research subject
Physics
Identifiers
URN: urn:nbn:se:kth:diva-144063ISBN: 978-91-7595-104-1 (print)OAI: oai:DiVA.org:kth-144063DiVA: diva2:710771
Public defence
2014-04-29, FA31, Roslagstullsbacken 21, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

QC 20140408

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2014-04-08Bibliographically approved
List of papers
1. Characterization of non-fluorescent transient states of tryptophan by fluorescence relaxation: Influence of buffers, additives and prospects for protein studies
Open this publication in new window or tab >>Characterization of non-fluorescent transient states of tryptophan by fluorescence relaxation: Influence of buffers, additives and prospects for protein studies
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Research subject
Physics
Identifiers
urn:nbn:se:kth:diva-144059 (URN)
Note

QS 2014

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2014-04-08Bibliographically approved
2. Dark states in ionic oligothiophene bioprobes-evidence from fluorescence correlation spectroscopy and dynamic light scattering
Open this publication in new window or tab >>Dark states in ionic oligothiophene bioprobes-evidence from fluorescence correlation spectroscopy and dynamic light scattering
Show others...
2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 22, 5924--5933 p.Article in journal (Refereed) Published
Abstract [en]

Luminescent conjugated polyelectrolytes (LCPs) can upon interaction with biological macromolecules change their luminescent properties, and thereby serve as conformation- and interaction-sensitive biomolecular probes. However, to exploit this in a more quantitative manner, there is a need to better understand the photophysical processes involved. We report studies of the conjugated pentameric oligothiophene, derivative p-FTAA, which changes optical properties with different p-FTAA concentrations in aqueous buffers, and in a pH and oxygen saturation dependent manner. Using dynamic light scattering, luminescence spectroscopy and fluorescence correlation spectroscopy, we find evidence for a monomer dimer equilibrium, for the formation of large clusters of p-FTAA in aqueous environment, and can couple aggregation to changed emission properties of oligothiophenes. In addition, we observe the presence of at least two dark transient states, one presumably being a triplet state. Oxygen was found to statically quench the p-FTAA fluorescence but also to promote molecular fluorescence by quenching dark transient states of the p-FTAA molecules. Taken together, this study provides knowledge of fluorescence and photophysical features essential for applying p-FTAA and other oligothiophene derivatives for diagnostic purposes, including detection and staining of amyloid aggregates.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2014
Keyword
FCS, FLUORESCENCE, CONJUGATED POLYMERS; POLYTHIOPHENE; DNA; AMYLOIDOSES; DERIVATIVES; ASSEMBLIES; SYSTEMS
National Category
Physical Chemistry
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-144060 (URN)10.1021/jp501324e (DOI)000337011400013 ()2-s2.0-84901982083 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2011.0218Swedish Research Council, VR-NT, 2012-3045
Note

Updated from "Manuscript" to "Published article". QC 20140707

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2017-12-05Bibliographically approved
3. Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
Open this publication in new window or tab >>Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-107520 (URN)
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QS 2012

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2016-05-27Bibliographically approved
4. Determination of molecular stoichiometry without reference samples by analysing fluorescence blinking with and without excitation synchronization
Open this publication in new window or tab >>Determination of molecular stoichiometry without reference samples by analysing fluorescence blinking with and without excitation synchronization
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Stoichiometry of molecular complexes plays a crucial role in biology. Moreover, for quantitative fluorescence studies, it is often useful to know the number of fluorophores labeled onto the molecules studied. In this work, we propose an approach to determine the number of independent fluorescence emitters on fluorescent molecules based on fluorescence blinking caused by photo-induced triplet state formation, photo-isomerization or charge transfer. The fluorescence blinking is measured under two different excitation regimes, on the same setup, and in one and the same sample. By comparing the fluorescence fluctuations under continuous excitation using Fluorescence Correlation Spectroscopy (FCS), when all the fluorophores are blinking independently of each other, with those occurring under square-pulsed excitation using Transient State (TRAST) spectroscopy, when all fluorophores are blinking in a synchronized manner, the number of fluorophores per molecule can be determined. No calibration sample is needed and the approach is independent of experimental conditions and of the specific environment of the molecules under study.

The approach was experimentally validated by labeling double stranded DNA (dsDNA) with different concentrations of the intercalating dye YOYO-1 Iodide. The sample was then measured consecutively by TRAST and FCS and the number of fluorophores per molecule was calculated. The determined numbers were found to agree well with the number of fluorophores per dsDNA, as determined from FCS measurements using additional calibration samples.

Keyword
FCS; transient state; stoichiometry; TRAST; photo-switching
National Category
Natural Sciences Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-144061 (URN)
Projects
VR-NT, 2012-3045
Funder
Swedish Research Council, VR-NT, 2012-3045Knut and Alice Wallenberg Foundation, 2011.0218
Note

QS 2014

Available from: 2014-04-08 Created: 2014-04-08 Last updated: 2016-03-10Bibliographically approved
5. Forster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores
Open this publication in new window or tab >>Forster Resonance Energy Transfer beyond 10 nm: Exploiting the Triplet State Kinetics of Organic Fluorophores
2011 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 45, 13360-13370 p.Article in journal (Refereed) Published
Abstract [en]

Inter- or intramolecular distances of biomolecules can be studied by Forster resonance energy transfer (FRET). For most FRET methods, the observable range of distances is limited to 1-10 nm, and the labeling efficiency has to be controlled carefully to obtain accurate distance determinations, especially for intensity-based methods. In this study, we exploit the triplet state of the acceptor fluorophore as a FRET readout using fluorescence correlation spectroscopy and transient state monitoring. The influence of donor fluorescence leaking into the acceptor channel is minimized by a novel suppression algorithm for spectral bleed-through, thereby tolerating a high excess (up to 100-fold) of donor-only labeled samples. The suppression algorithm and the high sensitivity of the triplet state to small changes in the fluorophore excitation rate make it possible to extend the observable range of FRET efficiencies by up to 50% in the presence of large donor-only populations. Given this increased range of FRET efficiencies, its compatibility with organic fluorophores, and the low requirements on the labeling efficiency and instrumentation, we foresee that this approach will be attractive for in vitro and in vivo FRET-based spectroscopy and imaging.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2011
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-50309 (URN)10.1021/jp206770s (DOI)000296686000029 ()2-s2.0-80855128935 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2009-3134EU, FP7, Seventh Framework Programme, 201 837
Note

QC 20150624

Available from: 2012-01-19 Created: 2011-12-05 Last updated: 2017-12-08Bibliographically approved

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