Related Experiment Video
Updated: May 28, 2026
Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)
Published on: April 6, 2012
Development of Lipid-Tethered Fluorescence Correlation Spectroscopy and Its Application to Conformational Dynamics of
Kaito Ogasawara1, Miyuki Sakaguchi1, Shoichi Yamaguchi1
1Department of Applied Chemistry, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama 338-8570, Japan.
Abstract:
Fluorescence correlation spectroscopy (FCS) is widely used to probe conformational dynamics of biomolecules; however, rapid diffusion in solution often obscures submillisecond to millisecond conformational transitions. Here, we introduce lipid-tethered FCS (LT-FCS), in which molecules are tethered to a cholesterol-rich supported lipid bilayer (SLB) to reduce lateral diffusion while maintaining molecular exchange in a focal region. The average residence time of molecules in the focal region is approximately 30-fold longer on the SLB than in solution, enabling clear separation of submillisecond conformational transition contributions in the autocorrelation function from the diffusion-related contributions. Furthermore, the use of fluorescence lifetime information on LT-FCS enables us to quantitatively analyze millisecond conformational transitions. We verified the capability of LT-FCS to analyze the submillisecond to millisecond conformational transition dynamics of hairpin DNAs with different stem lengths. LT-FCS thus provides a practical approach to access conformational dynamics in the submillisecond to millisecond regime, bridging the gap between conventional FCS and single-molecule FRET techniques.
