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
Analysis of Fluorescence Flicker as a Tool to Monitor Proton Transport and Biomolecular Interactions
KTH, School of Engineering Sciences (SCI), Physics.
2006 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The overall focus of this thesis is on fluorescence flicker processes of fluorescent molecules, e.g. protonation-deprotonation or singlet-triplet electronic state transitions, intrinsic or generated by their interaction with their environment, monitored by fluorescence spectroscopy.

Understanding proton migration along membranes and membrane proteins in cells is essential for understanding energy metabolism. It has been seen that certain membrane-spanning proton-transporter proteins in the respiratory chain in the mitochondrial inner membrane take up protons faster than the rate limited by diffusion. To explain these observations it has been suggested that there is a proton-collecting antenna, consisting of negatively and protonatable residues on the surface of these proteins, which increases the rate of uptake. Using fluorescence correlation spectroscopy and artificial biological membranes the proton collecting antenna effect is verified, as well as the proton migration properties on these membranes at various surface buffer concentrations.

Fluorescence flicker due to singlet-triplet electronic state transitions in a fluorescent molecule is interesting because of the long transition time between the two states. This means that the molecule has a long time to interact with the local environment, and can therefore be used as a microenvironmental sensor. A novel method for monitoring photo-induced, transient, long-lived, non- or weakly fluorescent states, e.g. the triplet state, was developed. With this method, only the time averaged intensity is detected and used for determining the triplet state kinetics. This method has several advantages, in particular it lends itself well for parallelization, over traditional methods including fluorescence correlation spectroscopy.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , iv, 30 p.
Series
Trita-FYS, ISSN 0280-316X ; 2006:75
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-4216ISBN: 978-91-7178-546-6 (print)OAI: oai:DiVA.org:kth-4216DiVA: diva2:11280
Presentation
2006-12-20, FA31, Albanova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00
Opponent
Supervisors
Note
QC 20101126Available from: 2006-12-07 Created: 2006-12-07 Last updated: 2010-11-26Bibliographically approved
List of papers
1. Localized Proton Microcircuits at the Biological Membrane-Water Interface
Open this publication in new window or tab >>Localized Proton Microcircuits at the Biological Membrane-Water Interface
2006 (English)In: PNAS, ISSN 0027-8424, Vol. 103, no 52, 19677-19770 p.Article in journal (Refereed) Published
Abstract [en]

Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters

Keyword
diffusion; fluorescence; membrane protein; pH; proton transfer
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-6539 (URN)10.1073/pnas.0605909103 (DOI)000243285500030 ()2-s2.0-33845936297 (Scopus ID)
Note

QC 20100809

Available from: 2006-12-07 Created: 2006-12-07 Last updated: 2016-05-16Bibliographically approved
2. Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Open this publication in new window or tab >>Monitoring Kinetics of Highly Environment Sensitive States of Fluorescent Molecules by Modulated Excitation and Time-Averaged Fluorescence Intensity Recording
Show others...
2007 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 79, no 9, 3330-3341 p.Article in journal (Refereed) Published
Abstract [en]

In this work, a concept is described for how the kinetics of photoinduced, transient, long-lived, nonfluorescent or weakly fluorescent states of fluorophore marker molecules can be extracted from the time-averaged fluorescence by using time-modulated excitation. The concept exploits the characteristic variation of the population of these states with the modulation parameters of the excitation and thereby circumvents the need for time resolution in the fluorescence detection. It combines the single-molecule sensitivity of fluorescence detection with the remarkable environmental responsiveness obtainable from long-lived transient states, yet does not in itself impose any constraints on the concentration or the fluorescence brightness of the sample molecules that can be measured. Modulation of the excitation can be performed by variation of the intensity of a stationary excitation beam in time or by repeated translations of a CW excitation beam with respect to the sample. As a first experimental verification of the approach, we have shown how the triplet-state parameters of the fluorophore rhodamine 6G in different aqueous enviroments can be extracted. We demonstrate that the concept is fully compatible with low time-resolution detection by a CCD camera. The concept opens for automated transient-state monitoring or imaging on a massively parallel scale and for high-throughput biomolecular screening as well as for more fundamental biomolecular studies. The concept should also be applicable to the monitoring of a range of other photoinduced nonfluorescent or weakly fluorescent transient states, from which subtle changes in the immediate microenvironment of the fluorophore marker molecules can be detected

Keyword
Biomarkers; CCD cameras; Imaging systems; Molecular biology; Sensitivity analysis
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-7194 (URN)10.1021/ac0622680 (DOI)000246027100008 ()2-s2.0-34248153327 (Scopus ID)
Note

QC 20100805

Available from: 2007-05-29 Created: 2007-05-29 Last updated: 2014-09-24Bibliographically approved

Open Access in DiVA

fulltext(22831 kB)345 downloads
File information
File name FULLTEXT01.pdfFile size 22831 kBChecksum MD5
92d14fa9598d7badaa4328564f73a97eac2446e62cff5d61af19b6c52d24bf29a6a8b488
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Sandén, Tor
By organisation
Physics
Physical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 345 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 425 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