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Scanning fluorescence correlation spectroscopy: a tool for probing microsecond dynamics of surface-bound fluorescent
Ying Xiao1, Volker Buschmann, Kenneth D Weston
1Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
Analytical Chemistry
|December 30, 2004
Summary
This study introduces a combined imaging and fluorescence correlation spectroscopy (FCS) method to analyze microsecond fluorescence dynamics. The technique characterizes dye kinetics and surface-bound molecule interactions, offering a rapid surface analysis tool.
Area of Science:
- Physical Chemistry
- Biophysics
- Spectroscopy
Background:
- Fluorescence correlation spectroscopy (FCS) is a powerful tool for studying molecular dynamics.
- Characterizing microsecond fluctuations in fluorescence emission is crucial for understanding photophysical processes and molecular interactions.
- Conventional FCS often requires specialized equipment and can be time-consuming for certain sample types.
Purpose of the Study:
- To develop and demonstrate a combined imaging and FCS method for characterizing microsecond fluorescence dynamics.
- To assess the method's ability to analyze the kinetics of fluorescent dyes transitioning to and from non-emitting states.
- To showcase the technique's utility for rapid characterization of surface-linked or thin-film samples.
Main Methods:
- A sample scanning laser confocal microscope was used with a low-cost counting board for 50 ns photon detection time resolution.
- Photon detection time data streams were utilized for both image generation and fluorescence signal fluctuation analysis.
- Correlation methods were applied to the photon data to extract dynamic information and species concentration/areal density.
Main Results:
- The combined method successfully characterized microsecond fluorescence fluctuations and dye kinetics (DiIC16, ATTO 520) transitioning to dark states.
- Results showed slight deviations from conventional models used for singlet-triplet conversion rates.
- The method also accurately determined the areal density of fluorescent species, analogous to concentration in solution-based FCS.
Conclusions:
- The developed surface-sensitive FCS method provides a rapid and convenient approach for characterizing surface-linked and thin-film samples.
- This technique can be applied to study photophysical phenomena, conformational changes, and interaction kinetics of immobilized species.
- The method holds potential for analyzing ligand-receptor interactions and biomolecular associations on microarrays.