Photobleaching Pathways in Single-Molecule FRET Experiments

Abstract

In order to acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons are needed. To achieve such fluxes one may use higher laser excitation intensity, however this induces increased rates of photobleaching. Anti-oxidants additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently-developed single molecule and ensemble spectroscopies, and doubly-labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways which place fundamental limitations on sm-FRET experiments. We find that: (i) acceptor photobleaching scales with FRET efficiency; (ii) acceptor photobleaching is enhanced under picosecond pulsed (vs. continuous wave) excitation; and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting using short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements.

Make them Blink: Probes for Super‐Resolution Microscopy

“…Empirically, we found that the excitation wavelength commonly determines the photobleaching pathway, that is, ROXS works best at longer excitation wavelength. [20,109,110] The generality of the concept is further underscored by the broad range of redox-active compounds including for example, ascorbic acid, trolox, mercaptoethylamine and ferrocene derivatives as reductants and MV, nitrobenzoic acid, 4-nitrobenzyl alcohol and troloxquinone as oxidants. [20,21,105,111] In this context, it is interesting to note that the antifading mechanism of trolox could be resolved studying the blinking of single oxazine molecules with functioned as quantitative sensors for reductants and oxidants (a single-molecule redox sensor).…”

Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)

“…8–15 However, smFRET has limited resolution of very fast events 27 and no capacity for long-duration monitoring of a single molecule. 28 For example, no smFRET measurement has measured more than three successive nucleotide incorporation events by the same KF molecule 9 . Rather than focusing on enzyme turnover, several smFRET experiments used a dideoxy substrate, which elegantly probes conformational dynamics during molecular recognition of the nucleotide.…”

Exploring one-state downhill protein folding in single molecules

“…The main issue is time resolution. Current SM-FRET dyes saturate at photon emissions of approximately 100 kHz (31), restricting the effective time resolution to the 0.5-1 ms it takes to detect statistically significant numbers of photons. We optimized the time-resolution/ photodamage trade-off by performing chemical-denaturation experiments at 279 K (which slows down BBL kinetics by approximately 200-fold) in conjunction with a newly developed photoprotection cocktail that affords emissions of approximately one photon per microsecond with little photodamage (32).…”