Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods

Sub-10nm gold nanoarrays for tethering single molecules

Nanometer sized gold patterns were produced with controlled spacings using the combination of a top-down (e-beam lithography) and a bottom-up (macromolecular chemistry) technique. Sub-10 nm nanoparticle arrays on silicon consisting of gold nanoparticles separated by micrometer spacings were fabricated with this approach. Using electron beam lithography, templates comprising of 150 nm to 1 mm sized trenches, holes and aperiodic patterns were made in an electron-beam resist. Block copolymer micelles were then patterned into this template by spincoating. The micelles acted as positioners for a nanometer sized gold precursor that is sequestered within its core. Subsequent removal of the resist layer left an array of Au loaded organic micelles ordered according to the pattern of the template. Exposure of this substrate to a hydrogen plasma removed the organic block copolymer and resulted in an array of sub-10 nm gold nanoparticles/nanoclusters with micron separations. The gold was then used as an anchor point for the tethering of functional molecules in order to localize fluorescent molecules

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack