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Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. (Experimental biomolecular physics)ORCID iD: 0000-0002-6191-9921
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorophore blinking dynamics are highly sensitive to the local environment and can be used as an additional readout parameter to increase the information gained from existing fluorescence techniques.The origin of these blinking patterns are photophysical transitions to and from a manifold of non-luminescent states. The long lifetime of these dark transient states, typically 103 to 106 times longer than the fluorescent state, gives them correspondingly more time to sense their environment. For this reason, fluorophore blinking dynamics are particularly sensitive to low frequency events, such as diffusion-mediated interactions between the fluorophore and dilute species.

Transient State (TRAST) monitoring has been developed to quantify fluorophore blinking dynamics in a simple and widely applicable manner. TRAST does not need to resolve individual blinking events, but instead monitors the average fluorescence intensity in response to a modulated excitation. By systematically varying the modulation parameters, the transient state kinetics of the sample are mapped out. Without the need for time-resolved detection, a regular camera can be used to image blinking dynamics with high spatial resolution.

This thesis presents TRAST characterizations of common autofluorescent compounds and demonstrates their ability to sense relevant biological parameters such as oxygen concentration and redox potential. In Papers I and II, the autofluorescent co-enzymes flavin and NAD(P)H were studied, and label-free imaging of local redox variations within cells was demonstrated. Perturbing the cells, through dilute additions of mitochondrial uncouplers, revealed a strong andlocalized response in the TRAST images. In Paper III we studied tryptophan autofluorescence and used it to detect conformational changes in an unlabeled spider silk protein.

Labeling with external fluorophores can add further specificity to the TRAST measurements. In Paper IV, TRAST was used to monitor diffusion-mediated interactions between lipids and receptors in a cell membrane, including the influence of receptor activation. In Paper V we tracked folding of RNA into G-quadruplexes in live cells, monitored via the isomerization properties of an attached cyanine dye.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. , p. i-vi; 116
Series
TRITA-SCI-FOU ; 2019:13
National Category
Other Physics Topics
Research subject
Biological Physics; Physics
Identifiers
URN: urn:nbn:se:kth:diva-246020ISBN: 978-91-7873-142-8 (print)OAI: oai:DiVA.org:kth-246020DiVA, id: diva2:1295462
Public defence
2019-04-05, FB53, KTH, Roslagstullsbacken 21, Stockholm, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-12 Created: 2019-03-11 Last updated: 2019-03-13Bibliographically approved
List of papers
1. Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy
Open this publication in new window or tab >>Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy
2018 (English)In: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 140, p. 178-187Article in journal (Refereed) Published
Abstract [en]

Transient state (TRAST) monitoring can determine population dynamics of long-lived, dark transient states of fluorescent molecules, detecting only the average fluorescence intensity from a sample, when subject to different excitation pulse trains. Like Fluorescence Correlation Spectroscopy (FCS), TRAST unites the detection sensitivity of fluorescence with the environmental sensitivity of long-lived non-fluorescent states, but does not rely on detection of stochastic fluorescence fluctuations from individual molecules. Relaxed requirements on noise suppression, detection quantum yield and time-resolution of the instrument, as well as on fluorescence brightness of the molecules studied, make TRAST broadly applicable, opening also for investigations based on less bright, auto-fluorescent molecules. In this work, we applied TRAST to study the transient state population dynamics within the auto-fluorescent coenzymes flavin adenine dinucleotide (FAD) and flavin-mononucleotide (FMN). From the experimental TRAST data, we defined state models, and determined rate parameters for triplet state and redox transitions within FMN and FAD, stacking and un-stacking rates of external redox active quenching agents and by the adenine moiety of FAD itself. TRAST experiments were found to be well capable to resolve these transitions in FMN and FAD, and to track how the transitions are influenced by ambient oxygenation and redox conditions. This work demonstrates that TRAST provides a useful tool to follow local oxygenation and redox conditions via FMN and FAD fluorescence, and forms the basis for measurements on flavoproteins and of redox and metabolic conditions in more complex environments, such as in live cells.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2018
Keywords
Flavin, Fluorescence, Triplet state, Redox state, Oxygenation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-232422 (URN)10.1016/j.ymeth.2017.11.013 (DOI)000436917700019 ()29179988 (PubMedID)2-s2.0-85036623291 (Scopus ID)
Funder
Swedish Research Council, VR-NT 2012-3045Swedish Foundation for Strategic Research Knut and Alice Wallenberg Foundation, KAW 2012.0218
Note

QC 20180725

Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2019-03-11Bibliographically approved
2. Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H
Open this publication in new window or tab >>Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). In contrast to FLIM, TRAST imaging of NAD(P)H clearly reveals an altered oxidative environment in the cytosols of cells treated with a mitochondrial un-coupler. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.

National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-246015 (URN)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-12Bibliographically approved
3. Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies
Open this publication in new window or tab >>Fluorescence-based characterization of non-fluorescent transient states of tryptophan - prospects for protein conformation and interaction studies
2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 35052Article in journal (Refereed) Published
Abstract [en]

Tryptophan fluorescence is extensively used for label-free protein characterization. Here, we show that by analyzing how the average tryptophan fluorescence intensity varies with excitation modulation, kinetics of tryptophan dark transient states can be determined in a simple, robust and reliable manner. Thereby, highly environment-, protein conformation- and interaction-sensitive information can be recorded, inaccessible via traditional protein fluorescence readouts. For verification, tryptophan transient state kinetics were determined under different environmental conditions, and compared to literature data. Conformational changes in a spider silk protein were monitored via the triplet state kinetics of its tryptophan residues, reflecting their exposure to an air-saturated aqueous solution. Moreover, tryptophan fluorescence anti-bunching was discovered, reflecting local pH and buffer conditions, previously observed only by ultrasensitive measurements in highly fluorescent photo-acids. Taken together, the presented approach, broadly applicable under biologically relevant conditions, has the potential to become a standard biophysical approach for protein conformation, interaction and microenvironment studies.

Place, publisher, year, edition, pages
Nature Publishing Group, 2016
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-196392 (URN)10.1038/srep35052 (DOI)000385352500001 ()2-s2.0-84994000069 (Scopus ID)
Note

QC 20161128

Available from: 2016-11-28 Created: 2016-11-14 Last updated: 2019-03-11Bibliographically approved
4. Transient state imaging of intermittent interactions between lipids and receptor proteins in artificial and live cell membranes
Open this publication in new window or tab >>Transient state imaging of intermittent interactions between lipids and receptor proteins in artificial and live cell membranes
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transient collisional interactions between lipids and membrane proteins play an important role in modulating cellular functions but occur at frequencies too low to be readily observable via fluorescence imaging or quenching studies. We used transient state imaging (TRAST) to quantify those interaction in living cells. This method combines sensitive detection of fluorescence from fluorophore marker molecules with the ability to monitor their long-lived dark triplet states, highly sensitive to molecular interactions in artificial and live cell membranes.

By TRAST we first determined the dark transient state kinetics of 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), an extensively used biomembrane fluorophore, available as a label on a wide range of lipids and sterols. We then measured quenching of NBD triplet states by spin-labels, in the membranes of small unilamellar vesicles (SUVs), and studied how it depends on the fluorescent lipid-derivative type and on the position of the spin label in the membranes. By the same strategy, we then quantified the collisional quenching of NBD-lipid derivatives and spin-labelled stearic acids in live cell plasma membranes.

Finally, we extended the method to study the collisional interactions between G-Protein Coupled Receptors (GPCRs), covalently labelled with the spin label TEMPO, and NBD-lipid derivatives, in the plasma membranes of living cells. Thereby, we could resolve transient interactions between the GPCRs and lipids with different hydrophilic heads or sterols, and how these interactions were changed upon activation of the GPCR by an agonist. The presented approach offers a straightforward and widely applicable means to characterize and image transient interactions in live cell membranes, of large biomedical relevance.

National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-246017 (URN)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

QC 20190312

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-12Bibliographically approved
5. Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of RNA G-quadruplexes in live cells
Open this publication in new window or tab >>Trans-cis isomerization kinetics of cyanine dyes reports on the folding states of RNA G-quadruplexes in live cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Guanine (G)-rich sequences in nucleic acids are prone to assemble into four-stranded structures, called G-quadruplexes. Abnormal GGGGCC repeat elongations have been associated with Amyotrophic lateral sclerosis and fronto-temporal dementia. In particular, the folding states of such elongations are believed to play a central role in the development of these diseases. Since most studies of G-quadruplex structures are made in vitro, it is highly relevant to clarify what the structures of elongated GGGGCC repeats look like in vivo. However, due to methodological constraints, evidence of specific structures of such GGGGCC repeats in vivo is sparse.

In this work, we devised a readout strategy, exploiting the sensitivity of trans-cis isomerization of cyanine dyes to local viscosity and sterical constraints. We show that the folding states of cyanine-labeled RNA molecules, and in particular of G-quadruplexes, can be identified in a sensitive manner by this strategy. The isomerization kinetics, monitored via the fluorescence blinking generated upon transitions between a fluorescent trans isomer and a non-fluorescent cis isomer, was first characterized for RNA molecules with GGGGCC repeats in aqueous solution using Fluorescence correlation spectroscopy (FCS) and transient state (TRAST) monitoring. With TRAST, monitoring the isomerization kinetics from how the average fluorescence intensity varies with modulation characteristics of a laser excitation source, we could then also detect the folding states of RNA molecules in living cells. This approach is robust, applicable on a broad range of biological samples and can also be extended to study folding or misfolding of proteins and biomolecules in general.

National Category
Other Physics Topics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-246018 (URN)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research
Note

QC 20190312

Available from: 2019-03-11 Created: 2019-03-11 Last updated: 2019-03-12Bibliographically approved

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