Single-molecule techniques eliminate ensemble averaging, thus revealing
transient or rare species in heterogeneous systems [1-3]. These
approaches have been employed to probe myriad biological phenomena,
including protein and RNA folding [4-6], enzyme kinetics [7, 8], and
even protein biosynthesis [1, 9, 10]. In particular,
immobilization-based fluorescence te- niques such as total internal
reflection fluorescence microscopy (TIRF-M) have recently allowed for
the observation of multiple events on the millis- onds to seconds
timescale [11-13]. Single-molecule fluorescence methods are challenged
by the instability of single fluorophores. The organic fluorophores
commonly employed in single-molecule studies of biological systems
display fast photobleaching, intensity fluctuations on the millisecond
timescale (blinking), or both. These phenomena limit observation time
and complicate the interpretation of fl- rescence fluctuations [14,
15]. Molecular oxygen (O) modulates dye stability. Triplet O
efficiently 2 2 quenches dye triplet states responsible for blinking.
This results in the for- tion of singlet oxygen [16-18]. Singlet O
reacts efficiently with organic dyes, 2 amino acids, and nucleobases
[19, 20]. Oxidized dyes are no longer fluor- cent; oxidative damage
impairs the folding and function of biomolecules. In the presence of
saturating dissolved O, blinking of fluorescent dyes is sup- 2 pressed,
but oxidative damage to dyes and biomolecules is rapid. Enzymatic O
-scavenging systems are commonly employed to ameliorate dye instability.
2 Small molecules are often employed to suppress blinking at low O
levels.
