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Assessment of the sensitivity of a confocal laser scanning microscope by fluorescence correlation spectroscopy and TRAST imaging
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.
Heinirich Heine University. (Chair for Molecular Physical Chemistry)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0003-3200-0374
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
National Category
Atom and Molecular Physics and Optics
Identifiers
URN: urn:nbn:se:kth:diva-105741OAI: oai:DiVA.org:kth-105741DiVA: diva2:571917
Note

QS 2012

Available from: 2012-11-26 Created: 2012-11-26 Last updated: 2013-01-07Bibliographically approved
In thesis
1. Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
Open this publication in new window or tab >>Förster Resonance Energy Transfer - from single molecule spectroscopy to imaging
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last fifteen years several methods have been developed for probing biomolecules (DNA, RNA, proteins) one at a time. Among these methods fluorescence spectroscopy and in particular its many implementations for monitoring Förster Resonance Energy Transfer (FRET), have attracted much interest.

This thesis deals mainly with high-precision single molecule FRET (smFRET) studies between a donor and an acceptor fluorophore attached to a biomolecule. Methodologies like multi-parameter fluorescence detection (MFD) and Probability Distribution Analysis (PDA) are used. We investigate, how and in which occasions; complex photophysical properties of the acceptor could influence the experimentally obtained FRET efficiency distributions. The value of smFRET experiments in enzymology is exemplified by presenting studies on DNA-related enzymes. Three structural conformations (Open, Closed, and Nucleotide-Binding) of Klentaq1, a DNA polymerase, have been resolved by measurements on freely diffusing molecules. We observe that the levels of occupancy of these conformations and the transitions among them, are dependent on the nature of the incoming dNTP, shedding more light into how conformational selection controls the incorporation cycle. Additionally, smFRET studies on MutS, a protein responsible for the initiation of the DNA mismatch repair machinery, have identified the existence of a preferred orientation of binding of the protein to asymmetric mismatches of DNA strands. Inhibiting MutS from binding in this preferred orientation has negative implications on the efficiency of the initiation of the overall DNA repair process.

Shifting from spectroscopy to microscopy, we use FRET imaging for monitoring interactions between the Human Epidermal Growth Receptors, HER1 and HER2, and the Insulin Growth Factor 1 Receptor, IGF1R, in fixed cells obtained from patients with suspect breast cancer lesions. While working on FRET imaging, the need for developing methodologies for the objective evaluation of the sensitivity of confocal laser scanning microscopes (CLSM) was identified. In order to provide figure of merits for the sensitivity of a microscope, we use Fluorescence Correlation Spectroscopy (FCS) and Transient State (TRAST) imaging measurements on aqueous solutions of Rhodamine 110. Our results suggest that TRAST imaging measurements could serve as a fast and easy test for the day-to-day maintenance of a CLSM and could provide reference standards for comparing images obtained by different microscope systems.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. 95 p.
Series
Trita-FYS, ISSN 0280-316X ; 2012:87
Keyword
FRET, FCS, single-molecule biophysics
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-105748 (URN)978-91-7501-575-0 (ISBN)
Public defence
2012-12-10, FA32, AlbaNova University Center, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20121126

Available from: 2013-04-02 Created: 2012-11-26 Last updated: 2013-04-02Bibliographically approved
2. 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

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