The project initially aimed to develop multimodal labelling probes for correlative nanoscale imaging by synthesizing functional fluorophores, lanthanide complexes, and hybrid nanobody–gold nanoparticle conjugates suitable for SIMS, STED, and electron microscopy. During the project, the required reagents and precursor materials were acquired, and multiple synthetic and bioconjugation strategies were systematically explored. Despite extensive optimization of reaction conditions based on established literature procedures and modified synthetic routes, the targeted probe systems could not be obtained with sufficient yield, stability, or reproducibility for downstream applications.
To ensure continued scientific progress, the research was redirected toward fundamental photophysical studies closely related to the original project objectives. Four well-defined donor–acceptor fluorophore pairs based on ATTO Rho6G and ATTO 647N connected by polyethylene glycol (PEG) linkers of different lengths were successfully designed and synthesized. These model systems enabled a systematic investigation of Förster resonance energy transfer (FRET) as a function of donor–acceptor separation, providing mechanistic insights relevant to the originally proposed dye–nanoparticle architectures.
The synthesized model compounds were comprehensively characterized using steady-state absorption and fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and femtosecond transient absorption spectroscopy. The measurements demonstrated the expected dependence of FRET efficiency on linker length in acetonitrile, whereas this trend was absent in aqueous solution, revealing an unexpected solvent-dependent behaviour that is currently under further investigation.
In parallel, the project contributed to a complementary study on the excited-state dynamics of the fluorescent pH probe pHrodo. Ultrafast transient absorption spectroscopy was employed to investigate the photophysical processes responsible for its pH-dependent fluorescence response, providing new mechanistic understanding of this widely used bioimaging probe.
Although the originally planned multimodal probes and imaging demonstrations could not be realized, the project generated valuable expertise in fluorophore synthesis, advanced optical spectroscopy, and mechanistic studies of excited-state dynamics. The work established robust experimental methodologies and produced reproducible datasets that form the basis of manuscripts currently in preparation.
