Fig. 3.

smFRET analysis of D135–L14 ribozymes. (A) Principle of total internal reflection fluorescence spectroscopy (TIRF). Molecules are immobilized on a quartz slide and placed in the microscope. The fluorophores are excited by the laser's evanescent wave above the critical angle, giving emission signals collected through the objective with a high-quantum yield camera. (B) smFRET time trace and histogram at 10 mM Mg²⁺ showing two conformational states at 0.25 and 0.4. (C) smFRET time trace and histogram at 100 mM Mg²⁺ sampling the three FRET states 0.25, 0.4, and 0.6. (D) (Left) smFRET time trace shows a dynamic behavior only upon increase in MgCl₂ concentration from 10 to 100 mM at 50–58 s. (Right) Distributions of the states before and after the addition of Mg²⁺.