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Transient State Monitoring by Total Internal Reflection Fluorescence Microscopy
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0002-5584-9170
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2010 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 11, 4035-4046 p.Article in journal (Refereed) Published
Abstract [en]

Triplet, photo-oxidized and other photoinduced, long-lived states Of fluorophores are sensitive to the local environment and thus attractive for microenvironmental imaging purposes. In this work, we introduce an approach where these states are monitored in a total internal reflection (TIR) fluorescence microscope, via the characteristic variations of the time-averaged fluorescence occuring ill response to different excitation modulation schemes. The surface-confined TIR excitation field generates a signal from the fluorescent molecules Close to the glass surface. Thereby, a high selectivity and low background noise is obtained, and in combination with IOW duty Cycles Of excitation, the overall photodegradation of the fluorescent molecules of the sample call be kept low, To verify the approach. the kinetics of the triplet and radical states of the dye Rhodamine 110 were imaged and analyzed in aqueous solutions at different concentrations of dissolved oxygen and of the reducing agent ascorbic acid. The experimental results Were compared to data from corresponding fluorescence correlation spectroscopy (FCS) measurements and simulations based oil finite element analysis. The approach was found to accurately determine relative populations and dynamics of triplet and photooxidized states, Overcoming passage time limitations seen ill FCS measurements. The method circumvents the need for time resolution ill the fluorescence detection, allowing simultaneous readout over the whole SLII-face area subject to excitation. It call be applied over a broad range of concentrations and does not require I strong fluorescence brightness of the sample molecules. Given the sensitivity of the triplet and photooxidized states to oxygen concentrations and not the least to local redox environments, we expect the approach to become an attractive tool for imaging cell metabolism.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2010. Vol. 114, no 11, 4035-4046 p.
Keyword [en]
single-molecule detection, spectroscopy tir-fcs, absorption-spectroscopy, dye molecules, phosphorescence, excitation, diffusion, transport, dynamics, oxygen
National Category
Physical Chemistry Biophysics
Research subject
Biological Physics
URN: urn:nbn:se:kth:diva-19319DOI: 10.1021/jp911034vISI: 000275710400036ScopusID: 2-s2.0-77949845445OAI: diva2:337366
Knut and Alice Wallenberg FoundationSwedish Research Council, VR-671-2006-3197EU, European Research Council, FLUODIA-201-837

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2016-03-10Bibliographically 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.
Trita-FYS, ISSN 0280-316X ; 2012:89
Transient States imaging (TRAST), Triplet State imaging, fluorescence microscopy, modulated excitation, triplet state, radical state, trans-cis isomerisation
National Category
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)
EU, FP7, Seventh Framework Programme, 201 837

QC 20130107

Available from: 2013-01-07 Created: 2012-12-27 Last updated: 2013-01-07Bibliographically approved

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