Blinking and its Effect on Single Molecule FRET Measurements

Abstract

A repeated cycle of fluorescence turning-on and -off or blinking behavior has been observed for single nanoparticles and green fluorescent protein (GFP) molecules. In this study, we found that chemical fluorescent dyes such as Cy5 and IC5 also show blinking. In single molecule FRET (smFRET) measurements using these fluorophores, the blinking effect should be taken into consideration, because the blinking of an acceptor results in apparent changes in the FRET efficiency. To distinguish the blinking from real FRET changes, the fluorescent state of an acceptor should be monitored at the same time. Donors (tetramethylrhodamine) and acceptors (IC5) were alternately excited by switching laser light between at 532 nm and 633 nm using electronic shutters. In addition, the donor and acceptor fluorescences were simultaneously imaged using a dual-view apparatus and recorded by an ICCD video camera at a video rate of 30 frames/sec. We found that some of smFRET efficiency changes were due to the blinking of the accepter. Thus, we could eliminate the blinking effect from smFRET measurements. After eliminating the blinking effect, we could analyze the FRET data from doubly labeled actin fixed on a glass surface.