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Updated: Jul 16, 2025

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules
Published on: April 25, 2025
A new twist on PIFE: photoisomerisation-related fluorescence enhancement
Evelyn Ploetz1, Benjamin Ambrose2,3, Anders Barth4
1Department of Chemistry and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Germany.
None:
PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.
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