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Inverse-Fluorescence Correlation Spectroscopy
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics.ORCID iD: 0000-0002-1850-5440
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.
2009 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 22, 9209-9215 p.Article in journal (Refereed) Published
Abstract [en]

An alternative version of fluorescence correlation spectroscopy is presented, where the signal from a medium surrounding the particles of interest is analyzed, as opposed to a signal from the particles themselves. Ibis allows for analysis of unlabeled particles and potentially of biomolecules. Here, the concept together with principal experiments on polystyrene beads of 100, 200, 400, and 800 nm diameter in an aqueous solution of alexa 488-fluorophores are presented. The use of photo detectors allowing higher photon fluxes, or of reduced detection volumes, should enable analysis of significantly smaller particles or even biomolecules.

Place, publisher, year, edition, pages
2009. Vol. 81, no 22, 9209-9215 p.
Keyword [en]
detection technology, diffusion
URN: urn:nbn:se:kth:diva-18945DOI: 10.1021/ac9010205ISI: 000271662400002ScopusID: 2-s2.0-70649097389OAI: diva2:336992
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2014-06-09Bibliographically approved
In thesis
1. Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
Open this publication in new window or tab >>Development and application of ultra-sensitive fluorescence spectroscopy and microscopy for biomolecular interaction studies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images.

Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xv, 79 p.
TRITA-FYS, ISSN 0280-316X ; 2014:23
single molecule spectroscopy, fluorescence correlation spectroscopy, stimulated emission microscopy, cancer, biomolecular interaction, co-localization
National Category
Physical Sciences
Research subject
Biological Physics
urn:nbn:se:kth:diva-146181 (URN)978-91-7595-180-5 (ISBN)
Public defence
2014-06-10, FB42, AlbaNova Universititetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)

QC 20140609

Available from: 2014-06-09 Created: 2014-06-09 Last updated: 2015-06-01Bibliographically approved

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Wennmalm, StefanThyberg, PerXu, LeiWidengren, Jerker
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