Freezing out structural effects: strong dye–dye couplings in gaseous rhodamine dimers at cryogenic temperatures

Förster resonance energy transfer (FRET) is a powerful distance-sensitive phenomenon that has been used to provide information on biomolecular conformations in the gas phase. At cryogenic temperatures, spectral narrowing enables selective excitation of specific conformers, expanding attainable structural insights. Here, we investigate rhodamine dimers connected by an alkynyl linker, where each dye is separated from the linker by a methylene group to break the conjugation with the dye, at <100 K. Rhodamine dyes are highly sensitive to their local environment, including proximal π-systems, so a detailed understanding of their intrinsic photophysics at low temperatures is essential. Using the LUNA2 setup in Aarhus, we recorded absorption and fluorescence spectra from mass-selected gaseous ions stored in a cryogenic ion trap. Cold rhodamine monomers attached to the linker show large redshifts, consistent with polarisation induced by the nearby π-system. Homodimers exhibit dramatic redshifts compared to both bare dyes and room-temperature measurements of identical species, suggesting compact geometries at low temperature, supported by DFT calculations. TD-DFT results likewise predict redshifts both from the π-system and dye-dye proximity. The magnitude of these shifts suggests excitonic coupling, consistent with calculated dye-dye separations of <5 Å. For heterodimers, nearly complete FRET is observed, and selective excitation of distinct absorption bands reveals conformer-specific emission, that is, it is possible to target specific conformers without prior selection. Together, these findings advance understanding of dye-dye interactions in cold environments and reinforce cryo-FRET's effectiveness at probing biomolecular structure.