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Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. (Experimental Biomolecular Physics)ORCID iD: 0000-0002-6191-9921
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. (Experimental Biomolecular Physics)
Institute of Physics, University of Belgrade.
KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics. (Experimental Biomolecular Physics)ORCID iD: 0000-0003-3200-0374
(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: urn:nbn:se:kth:diva-246015OAI: oai:DiVA.org:kth-246015DiVA, id: diva2:1295284
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
In thesis
1. Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
Open this publication in new window or tab >>Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
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:nbn:se:kth:diva-246020 (URN)978-91-7873-142-8 (ISBN)
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

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