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Trans-cis isomerization of lipophilic dyes provides a measure of membrane microviscosity in biological membranes and in live cells
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-107520OAI: oai:DiVA.org:kth-107520DiVA: diva2:580783
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
In thesis
1. Transient State Fluorescence Microscopy - method development and biological applications: Exploiting the dark states of fluorophores to measure oxygen concentrations, redox state, Förster resonance energy transfer and membrane viscosity
Open this publication in new window or tab >>Transient State Fluorescence Microscopy - method development and biological applications: Exploiting the dark states of fluorophores to measure oxygen concentrations, redox state, Förster resonance energy transfer and membrane viscosity
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Due to their long lifetime, triplet, redox and other transient states of fluorophores are highly sensitive to the micro-environment. Imaging their spatial distribution in biological samples can therefore help answer interesting questions about the metabolism, molecular interactions and dynamics in living cells. However, as these states are at best weakly luminescent, they have up to now only been used to a limited extent in life sciences. In Transient State (TRAST) imaging, the characteristic build up of transient states is instead monitored via fluorescence, as the excitation is modulated. When the illumination pulse width is step-wise increased, transient states are progressively populated. The resulting depletion of the singlet excited state can be monitored via time-averaged fluorescence. This fluorescence decay is characteristic for the transient state kinetics of the fluorophore in a given environment. Traditional fluorescence parameters can only be influenced within the lifetime of the fluorophore. In contrast, TRAST imaging can monitor photo-induced states with 103− 106 times longer lifetimes and is therefore far more sensitive to sparse quencher molecules, such as dissolved oxygen. Transient state kinetics can also be studied using Fluorescence Correlation Spectroscopy (FCS). In contrast to FCS, transient state imaging circumvents the need of time resolution in the fluorescence detection, thereby allowing simultaneous readout over a large number of pixels using a camera. It can also be applied over a broader range of concentrations and does not require a strong fluorescence brightness of the sample molecules. In this thesis, TRAST imaging has been applied in a total internal reflection fluorescence microscope to monitor the redox reactions of fluorescent dyes in solution. Moreover, TRAST imaging was established for measuring lipid microfluidity in biomembranes, and for a new concept to measure molecular distances in combination with Förster Resonance Energy Transfer. The sensitivity of the fluorophore triplet state to oxygen has been exploited in a wide-field microscope to monitor oxygen consumption during the contraction of smooth muscle cells and the modulation of the oxygen consumption of cancer cells by metabolite availability. High triplet yield fluorophores such as Eosin Y are introduced in order to reduce irradiance intensity requirements as reported in earlier TRAST papers. Irradiance requirements and axial resolution have further been reduced using a single plane illumination microscope.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. xiii, 94 p.
Series
Trita-FYS, ISSN 0280-316X ; 2012:89
Keyword
Transient States imaging (TRAST), Triplet State imaging, fluorescence microscopy, modulated excitation, triplet state, radical state, trans-cis isomerisation
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-109278 (URN)978-91-7501-608-5 (ISBN)
Public defence
2013-01-11, Sal FA31, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QC 20130107

Available from: 2013-01-07 Created: 2012-12-27 Last updated: 2013-01-07Bibliographically approved
2. Fluorescence-based methods to probe long-lived transient states for biomolecular studies
Open this publication in new window or tab >>Fluorescence-based methods to probe long-lived transient states for biomolecular studies
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:nbn:se:kth:diva-144063 (URN)978-91-7595-104-1 (ISBN)
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
3. Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Open this publication in new window or tab >>Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. x, 59 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2016:22
Keyword
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-187708 (URN)978-91-7729-026-1 (ISBN)
Public defence
2016-06-13, FB52, KTH, AlbaNova University Center, Roslagsvägen 30 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-26 Last updated: 2016-05-31Bibliographically approved

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