Force–Forster Resonance Energy Transfer Correlation Microscopy: A Multimodal Approach for the Study of Single‐Nucleotide‐Resolution Ribosome Translocation

Force spectroscopy and Förster resonance energy transfer (FRET) are both widely applicable techniques for biomolecular research. Each has its own advantages and also limitations, such as the interference of nonspecific binding for force spectroscopy and difficulty in obtaining single-nucleotide resolution for FRET. Here, we show a multimodal method of force-FRET correlation (FFC) microscopy to offer single-nucleotide resolution at single-molecule level while eliminating the interference of nonspecific interactions. This technique uses both magnetic and fluorescent labeling and employs two mechanical forces with different forms and amplitudes. Molecular specificity was achieved via the combination of removing nonspecific interactions and establishing force-FRET colocalization. High molecular resolution was obtained by applying precisely controlled acoustic forces on the intermolecular bonds. We have applied FFC microscopy for ribosome translocation study to demonstrate its capability for investigating complex biological functions. With high molecular resolution and straightforward implementation, our technique has the potential of broad applications in biochemistry and biophysics.