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Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes
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.
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2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 11, 3821-3830 p.Article in journal (Refereed) Published
Abstract [en]

An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured

Place, publisher, year, edition, pages
2010. Vol. 99, no 11, 3821-3830 p.
Keyword [en]
National Category
Research subject
Biological Physics
URN: urn:nbn:se:kth:diva-27983DOI: 10.1016/j.bpj.2010.09.059ISI: 000285033800035ScopusID: 2-s2.0-78649894488OAI: diva2:382695
EU, European Research Council, 201 837Swedish Research Council, VR-NT 2009-3134Knut and Alice Wallenberg Foundation

QC 20110103

Available from: 2011-01-03 Created: 2011-01-03 Last updated: 2016-03-10Bibliographically approved
In thesis
1. Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
Open this publication in new window or tab >>Fluorescence Studies of Membranes -- Proteins and Lipids, their Dynamics and Interactions
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, fluorescence spectroscopy was utilized to probe protein and lipid dynamics and interactions in their native, or close to native, environments. Thereby insight could be gained into the fundamentals of bacterial cell division and the innateimmune system.

A particular focus has been devoted to fluorescence fluctuations. They arise when a lower number of fluorescent molecules undergo Brownian motion through a confocal detection volume. With sensitive detectors, appropriate optics and efficient data acquisition these fluctuations can be observed and correlated. This is the heart in fluorescence correlation spectroscopy (FCS), conceived in the 1970’s. FCS has the power to quantify absolute concentrations, diffusion coefficients and to some extent binding events, and is suitable for measurements on a range of samples, including living cells.

However, FCS is not limited to solely diffusional processes. The sensitivity and time resolution allows also electron transitions within the fluorescent molecules to be probed. Of particular interest are spin transitions to and from the dark triplet state. This state is long-lived and sensitive, making it an effective sensor of the surrounding environment. We found that the triplet state could also be used to probe low frequency interactions in membranes down to a single molecule level and a theoretical model was developed that supported the observed interactions. FCS can be extended to fluorescence cross-correlation (FCCS), to handle also a second type of fluorescent molecule, fluorescing with a different colour, and whose signal is cross-correlated with the signal from the first type of fluorescent molecules. The aim was to improve and apply FCCS as a screening tool to probe proteinprotein interactions. By utilizing a methodology based on fluorescently labelled antibodies, we were able to calibrate our FCCS system and provide quantitative data on a particular receptor interaction occurring in natural killer cells. The methodology was found to increase the possibility to quantitatively analyse protein-protein interactions in the membrane of living cells.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 55 p.
Trita-FYS, ISSN 0280-316X ; 22
Fluorescence correlation spectroscopy, cytokinesis, collision theory, protein-protein interactions, lipid dynamics, single-molecule experiment
National Category
Condensed Matter Physics
urn:nbn:se:kth:diva-33842 (URN)978-91-7501-027-4 (ISBN)
Public defence
2011-06-01, FA32, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00 (English)
QC 20110523Available from: 2011-05-23 Created: 2011-05-19 Last updated: 2011-05-23Bibliographically approved

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Strömqvist, JohanChmyrov, AndriyJohansson, SofiaAndersson, AugustWidengren, Jerker
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