Fig. 3: Illumination-intensity-dependent changes in donor and acceptor brightness.
From: Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation
a, Representative single-molecule donor and acceptor fluorescence traces used for brightness calculations, which are crosstalk and acceptor direct excitation corrected, but not γ-corrected. \({{{I}}}_{\rm{D}}^{\;{\rm{FRET}} }\) and \({{{I}}}_{\rm{A}}^{\;{\rm{FRET}} }\) indicate brightness of the donor and acceptor fluorophore, respectively, during a FRET process. \({{{I}}}_{\rm{D}}^{{\rm{No}}\;{\rm{FRET}}}\) indicates brightness of donor fluorophore after acceptor photobleaching. b,c, Total (donor + acceptor) intensity of the single-molecule trace before (b) and after (c) γ correction. d,e, Changes of γ-uncorrected (d) donor and (e) acceptor brightness (photon counts per 100 ms frames) for Cy3–Cy5 and LD555–LD655 FRET pairs attached to the DNA duplex (Fig. 2a) with increasing illumination intensity. Data were collected in deoxygenated imaging buffers in the absence of any exogenous solution additives (photoprotective agents) at 100 ms time resolution using a custom-built TIRF imaging platform51. Error bars represent the s.d. of mean intensity values from five experimental repeats. f, Variations of empirical γ correction parameter of Cy3–LD655, Cy3–Cy5, LD555–LD655 and LD555–Cy5-labeled DNA oligonucleotides imaged in deoxygenated imaging buffers in the absence of any exogenous solution additives at 100 ms time resolution at increasing illumination intensity. Error bars for Cy3–Cy5 and LD555–LD655 data represent the s.d. of mean γ values from five experimental repeats. Error bars for Cy3–LD655 and LD555–Cy5 data represent the s.d. of mean γ values from three experimental repeats.
