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Scanning inverse fluorescence correlation spectroscopy
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.ORCID iD: 0000-0003-3200-0374
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-1850-5440
2014 (English)In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 22, no 11, p. 13073-13090Article in journal (Refereed) Published
Abstract [en]

Scanning Inverse Fluorescence Correlation Spectroscopy (siFCS) is introduced to determine the absolute size of nanodomains on surfaces. We describe here equations for obtaining the domain size from cross-and auto-correlation functions, measurement simulations which enabled testing of these equations, and measurements on model surfaces mimicking membranes containing nanodomains. Using a confocal microscope of 270 nm resolution the size of 250 nm domains were estimated by siFCS to 257 +/- 12 nm diameter, and 40 nm domains were estimated to 65 +/- 26 nm diameter. Applications of siFCS for sizing of nanodomains and protein clusters in cell membranes are discussed.

Place, publisher, year, edition, pages
Optical Society of America, 2014. Vol. 22, no 11, p. 13073-13090
Keywords [en]
Image Correlation Spectroscopy, Cross-Correlation Spectroscopy, Stimulated-Emission, Microscopy, Nanoscopy, Proteins, Cells, Limit
National Category
Biophysics
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-148302DOI: 10.1364/OE.22.013073ISI: 000337501600037Scopus ID: 2-s2.0-84901830506OAI: oai:DiVA.org:kth-148302DiVA, id: diva2:736674
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140808

Available from: 2014-08-08 Created: 2014-08-05 Last updated: 2019-04-04Bibliographically approved
In thesis
1. Super resolution fluorescence imaging: analyses, simulations and applications
Open this publication in new window or tab >>Super resolution fluorescence imaging: analyses, simulations and applications
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 81
Series
TRITA-SCI-FOU ; 2019:20
National Category
Other Physics Topics
Research subject
Physics
Identifiers
urn:nbn:se:kth:diva-248297 (URN)978-91-7873-171-8 (ISBN)
Public defence
2019-04-26, FA32, KTH, Roslagstullsbacken 21, Stockholm, 18:22 (English)
Opponent
Supervisors
Note

QC 20190405

Available from: 2019-04-05 Created: 2019-04-04 Last updated: 2019-04-05Bibliographically approved

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Bergstrand et al Opt Expr 2014(892 kB)129 downloads
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Bergstrand, JanRönnlund, DanielWidengren, JerkerWennmalm, Stefan
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