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Modulation Filtering Enables Removal of Spikes in Fluorescence Correlation Spectroscopy Measurements without Affecting the Temporal Information
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimental Biomolecular Physics)
KTH, School of Engineering Sciences (SCI), Applied Physics. (Experimental Biomolecular Physics)ORCID iD: 0000-0003-3200-0374
2009 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 113, no 25, 8752-8757 p.Article in journal (Refereed) Published
Abstract [en]

The appearance of intensity spikes in measurements is a common problem in fluorescence correlation spectroscopy (FCS) studies of biological samples. In this work, we present a new method for generating artifact-free correlation curves from fluorescence traces that have undergone spike removal. This method preserves the temporal information throughout the measurement and properly represents the correlation between events separated by removed spikes. The method was validated using experimental data. The proposed algorithm is demonstrated herein to be generally applicable, but it is particularly powerful for cases where spikes occur frequently.

Place, publisher, year, edition, pages
2009. Vol. 113, no 25, 8752-8757 p.
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-10238DOI: 10.1021/jp902538bISI: 000267205600045Scopus ID: 2-s2.0-67649218418OAI: oai:DiVA.org:kth-10238DiVA: diva2:212171
Note
QC 20100805Available from: 2009-04-21 Created: 2009-04-21 Last updated: 2010-12-06Bibliographically approved
In thesis
1. Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications
Open this publication in new window or tab >>Temporal Modulation in Fluorescence Spectroscopy and Imaging for Biological Applications
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis explores the benefits of intensity modulation for the purpose of extending the range of applications of fluorescence spectroscopy and imaging in cellular and molecular biology and medicine.

Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the detection sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and wide-field imaging possible developments. An extension of this method, generating image contrast based on triplet state population using a standard laser scanning microscope, is also shown.

A strategy to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize measurement conditions with respect to photophysical properties of the dyes used. FCS with modulated excitation will probably prove useful in future studies involving multiple kinetic processes occurring in overlapping time ranges. One of the ideas from this project also constitutes a powerful method for generating artifact free correlation curves from data sets where sections have been removed. This is potentially very useful in biological studies where spikes in the measurements often cause problems.

In the final project, cross-correlation and alternating excitation are combined in measurements on a pH-sensitive ratiometric dye to clearly distinguish the protonation–deprotonation dynamics from other processes. The presented approach makes the protonation related fluctuations manifest themselves as a very distinct anti-correlating component in the correlation curve. This enables robust data analysis using a simple model.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 81 p.
Series
Trita-FYS, ISSN 0280-316X ; 2009:13
Keyword
fluorescence, spectroscopy, microscopy, modulated excitation, intensity modulation, fluorescence correlation spectroscopy, transient states, molecular kinetics
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-10243 (URN)978-91-7415-299-9 (ISBN)
Public defence
2009-05-20, FB42, AlbaNova main building, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20100805Available from: 2009-05-12 Created: 2009-04-21 Last updated: 2010-08-05Bibliographically approved

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