SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Modulated Fluorescence Correlation Spectroscopy with complete time range information
Gustav Persson 1, Per Thyberg 1 and Jerker Widengren 1*
1 KTH (Royal Institute of Technology)
* To whom correspondence should be addressed. E-mail: jerker{at}biomolphysics.kth.se.
Submitted on May 28, 2007
Revised on June 29, 2007
Accepted on 13 September 2007
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Abstract |
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Two methods to combine fluorescence correlation spectroscopy (FCS) with modulated excitation, in a way that allows extraction of correlation data for all correlation times have been developed and experimentally verified. One method extracts distorsion-free correlation data from measurements acquired with standard hardware correlators provided the fluorescence does not change systematically within the excitation pulses. This restriction does not apply to the second method, which however requires acquisition of the raw fluorescence intensity. Modulation of the excitation in an FCS experiment is demonstrated to suppress triplet population build-up more efficiently than a corresponding reduction in continuous wave excitation intensity (shown for the dye rhodamine 6G in aqueous solution). Excitation modulation thus offers an additional means to optimize the FCS measurement conditions with respect to the photophysical properties of the dyes used. This possibility to suppress photo-induced states also provides a useful tool to distinguish additional processes occurring in the same time regime in the FCS measurements, as demonstrated here for the protonation kinetics of fluorescein at different pH. In general, the proposed method opens for FCS measurements with a complete correlation time scale in a range of applications where a modulated excitation is either necessary or brings specific advantages.
Key Words:
FCS, fluorescence, modulation, triplet state
