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Label-free monitoring of ambient oxygenation and redox conditions using the photodynamics of flavin compounds and transient state (TRAST) spectroscopy
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Kvant- och biofotonik.ORCID-id: 0000-0002-6191-9921
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Kvant- och biofotonik.ORCID-id: 0000-0003-3200-0374
2018 (Engelska)Ingår i: Methods, ISSN 1046-2023, E-ISSN 1095-9130, Vol. 140, s. 178-187Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
ACADEMIC PRESS INC ELSEVIER SCIENCE , 2018. Vol. 140, s. 178-187
Nyckelord [en]
Flavin, Fluorescence, Triplet state, Redox state, Oxygenation
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-232422DOI: 10.1016/j.ymeth.2017.11.013ISI: 000436917700019PubMedID: 29179988Scopus ID: 2-s2.0-85036623291OAI: oai:DiVA.org:kth-232422DiVA, id: diva2:1235357
Forskningsfinansiär
Vetenskapsrådet, VR-NT 2012-3045Stiftelsen för strategisk forskning (SSF)Knut och Alice Wallenbergs Stiftelse, KAW 2012.0218
Anmärkning

QC 20180725

Tillgänglig från: 2018-07-25 Skapad: 2018-07-25 Senast uppdaterad: 2019-03-11Bibliografiskt granskad
Ingår i avhandling
1. Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
Öppna denna publikation i ny flik eller fönster >>Fluorescence-based Transient State Monitoring for biomolecular, cellular and label-free studies
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
KTH Royal Institute of Technology, 2019. s. i-vi; 116
Serie
TRITA-SCI-FOU ; 2019:13
Nationell ämneskategori
Annan fysik
Forskningsämne
Biologisk fysik; Fysik
Identifikatorer
urn:nbn:se:kth:diva-246020 (URN)978-91-7873-142-8 (ISBN)
Disputation
2019-04-05, FB53, KTH, Roslagstullsbacken 21, Stockholm, 09:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
VetenskapsrådetCancerfondenStiftelsen för strategisk forskning (SSF)Knut och Alice Wallenbergs Stiftelse
Anmärkning

QC 20190312

Tillgänglig från: 2019-03-12 Skapad: 2019-03-11 Senast uppdaterad: 2019-03-13Bibliografiskt granskad

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