Single-walled carbon nanotubes (SWNTs) have unique photophysical properties but low fluorescence efficiency. We have found significant increases in the fluorescence efficiency of individual DNA-wrapped SWNTs upon addition of reducing agents, including dithiothreitol, Trolox, and β-mercaptoethanol. Brightening was reversible upon removal of the reducing molecules, suggesting that a transient reduction of defect sites on the SWNT sidewall causes the effect. These results imply that SWNTs are intrinsically bright emitters and that their poor emission arises from defective nanotubes.

We demonstrate that Zn(II) porphyrin in Zn(II)cytochrome c (Zn cyt c) is a fluorescence resonance energy transfer (FRET) donor to an Alexa660 dye acceptor. The energy transfer efficiency is dependent on the distance between the two fluorophores as shown through protein denaturation studies of five Zn cyt c variants labeled with Alexa660 in different positions. The relative quantum yield, excitation and emission energies, and labeling efficiencies of this donor-acceptor pair allow for a method of analysis based on sensitized emission of the acceptor. These studies show that Zn(II) porphyrin is an effective energy donor for measurement of molecular-scale distances by FRET.

Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochrome c molecules. In particular, measurements of the fluorescence lifetimes of individual cytochrome c molecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly-labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single-proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity.