Abstract
Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET’s broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.
Highlights
Single-molecule Förster Resonance Energy Transfer is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules
Two experimental formats are commonly employed to obtain Single-molecule Förster Resonance Energy Transfer (smFRET) data: a confocal approach, in which individual molecules are detected as they diffuse through a confocal volume[3]; and a total internal reflection fluorescence (TIRF) microscopy approach[12], in which individual molecules are immobilised on a glass coverslip and excited by an evanescence field
Here we provide detailed build instructions, parts lists, and open-source acquisition software, to enable a broad range of scientists to perform confocal smFRET experiments, on a validated, self-built, robust and economic instrument
Summary
We present the smfBox[18], a cost-effective confocal-based platform capable of measuring the FRET efficiency between dye pairs on freely diffusing single molecules, using variable alternating laser excitation (ALEX)[19] for verification of correct dye stoichiometry and the determination of accurate FRET correction factors[5,20,21]. Using the published correction procedures implemented in our open-source python analysis (Jupyter notebooks - Supplementary Note 6), we obtained data in excellent agreement with those from the other labs in the blind study This provides both an excellent validation of the smfBox, and a useful diagnostic for users to test their own builds of this instrument, as the successful reproduction of these data means that all hardware, acquisition and analysis software must be working correctly. In cases where the static (low-FRET and high-FRET) species have considerable overlap with each other (and with the dynamic population) the dPDA model is less well constrained, leading to a greater variation in the values of recovered rates for a given sample size These results have implications for the optimal positioning of FRET dyes when designing dynamic experiments
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