Towards cryogenic single-molecule FRET spectroscopy as a structural biology tool

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

Conducting cryo-smFRET requires solving a critical technical challenge first: the “dipole orientations” of the donor and acceptors dyes need to be measured when the sample is frozen to infer distance (which is not a problem for room-temperature in solution smFRET because the orientations average out due to rapid rotational dynamics of the probes). We propose a solution to this problem by adapting an unconventional, “dual-view”, imaging geometry to estimate both the absorption and emission dipole orientations of single emitters. We further evaluate the performance of the proposed scheme using physical modeling and numerical simulations. The results show that our new scheme achieves unparallel precision and flexibility in estimating the absorption dipole orientations and is also capable of simultaneous estimation of emission dipole orientations. This work paves the way to ultimately realizing cryo-smFRET spectroscopy. A preprint on this novel concept and simulation validation has been posted (https://www.biorxiv.org/content/10.1101/2025.06.17.660171v1) and the paper is in revision as of Sept. 2025.