Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Triplet Imaging of Oxygen Consumption during the Contraction of a Single Smooth Muscle Cell (A7r5)
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.
Vise andre og tillknytning
2010 (engelsk)Inngår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, nr 2, s. 339-349Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

sted, utgiver, år, opplag, sider
2010. Vol. 98, nr 2, s. 339-349
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-28879DOI: 10.1016/j.bpj.2009.10.006ISI: 000273972700018Scopus ID: 2-s2.0-77049088951OAI: oai:DiVA.org:kth-28879DiVA, id: diva2:395541
Merknad
QC 20110207Tilgjengelig fra: 2011-02-07 Laget: 2011-01-21 Sist oppdatert: 2017-12-11bibliografisk kontrollert
Inngår i avhandling
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
Åpne denne publikasjonen i ny fane eller vindu >>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 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2012. s. xiii, 94
Serie
Trita-FYS, ISSN 0280-316X ; 2012:89
Emneord
Transient States imaging (TRAST), Triplet State imaging, fluorescence microscopy, modulated excitation, triplet state, radical state, trans-cis isomerisation
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-109278 (URN)978-91-7501-608-5 (ISBN)
Disputas
2013-01-11, Sal FA31, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, 201 837
Merknad

QC 20130107

Tilgjengelig fra: 2013-01-07 Laget: 2012-12-27 Sist oppdatert: 2013-01-07bibliografisk kontrollert

Open Access i DiVA

Fulltekst mangler i DiVA

Andre lenker

Forlagets fulltekstScopus

Søk i DiVA

Av forfatter/redaktør
Spielmann, Thiemo
Av organisasjonen
I samme tidsskrift
Biophysical Journal

Søk utenfor DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric

doi
urn-nbn
Totalt: 188 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf