Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Transient State Imaging by Single Plane Illumination Microscopy of MCF-7 cells
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.
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics
Identifiers
URN: urn:nbn:se:kth:diva-107518OAI: oai:DiVA.org:kth-107518DiVA: diva2:580782
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

NQC 2015

Available from: 2012-12-27 Created: 2012-12-12 Last updated: 2015-04-27Bibliographically 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.
Series
Trita-FYS, ISSN 0280-316X ; 2012:89
Keyword
Transient States imaging (TRAST), Triplet State imaging, fluorescence microscopy, modulated excitation, triplet state, radical state, trans-cis isomerisation
National Category
Biophysics
Identifiers
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)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 201 837
Note

QC 20130107

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

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Mücksch, JonasSpielmann, ThiemoSisamakis, Evangelos
By organisation
Experimental Biomolecular Physics
Biophysics

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 189 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf