Alex Single Molecule Three Color in TIRF Microscopy

Abstract

Single-molecule three-color FRET is powerful in studying complex molecular interactions. Existing techniques, however, have been limitedly used for immobilized molecules, and never realized in TIRF microscopy. The main reason of this limited utilization is poor photostability of FRET probes selected for single-molecule three-color FRET. In this work, we realized single-molecule three-color FRET in TIRF microscope for the first time. By using Cy3, Cy5, and Cy7 as FRET probes, we could use the conventional oxygen scavenger system with Trolox to efficiently reduce photobleaching of all fluorophores. Well-separated emission peaks of three fluorophores made data analysis more reliable. To monitor three-domain motion in real time, we synchronized the data acquisition of EM-CCD with the fast switching of 532-nm and 633-nm lasers. To demonstrate the capability of the setup, we observed the conformational dynamics of the Holliday junction and that of RNA 4-way junction from the hairpin ribozyme.