Multimodal labelling probes for correlative nanoscale mass spectroscopy imaging, optical and electron microscopies

Project Information

MultiModLabPro

Grant agreement ID: 101107611

DOI

10.3030/101107611

Project closed

EC signature date 10 March 2023

Start date 15 March 2024

End date 14 March 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 206 887,68

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - MultiModLabPro (Multimodal labelling probes for correlative nanoscale mass spectroscopy imaging, optical and electron microscopies)

Reporting period: 2024-03-15 to 2026-03-14

Summary of the context and overall objectives of the project

The project aimed to develop advanced fluorescent probe systems for correlative and multimodal imaging by integrating organic fluorophores, metal coordination chemistry, and bioconjugation strategies. The original concept focused on the synthesis of polyhalogenated rhodamine dyes, their functionalization via DOTA–lanthanide coordination chemistry, and subsequent conjugation to gold nanoparticles and nanobodies for applications in cellular imaging.
Due to unforeseen synthetic challenges in the preparation of the targeted probe systems, the project was partially redirected toward mechanistic photophysical studies using simplified model systems. In particular, Förster resonance energy transfer (FRET) between donor (Rho6G) and acceptor (647N) fluorophores connected via PEG linkers of varying lengths was investigated as a surrogate system to study distance-dependent energy transfer processes relevant to nanoparticle–dye assemblies. In parallel, the excited-state dynamics of a pH-responsive fluorophore (pHrodo) were studied to gain insight into ultrafast photophysical behaviour of rhodamine-based systems.
The project was carried out between the University of Gothenburg and the University of Copenhagen, with the majority of experimental work performed at UCPH using advanced steady-state and ultrafast spectroscopic techniques.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Although the originally proposed multimodal imaging probes were not successfully realized, the project produced scientifically valuable results that contribute to the understanding of photophysical processes in fluorescent molecular systems. The development of well-defined donor–acceptor model compounds enabled systematic studies of FRET using complementary steady-state, time-resolved, and ultrafast spectroscopic techniques. The observation of pronounced solvent-dependent FRET behaviour provides new insights into the influence of the local environment on energy transfer, extending current knowledge beyond conventional distance-dependent descriptions.
The project also generated new mechanistic information on the excited-state dynamics of the pH-sensitive fluorophore pHrodo, contributing to a better understanding of the operation of fluorescent probes widely used in biological imaging. These findings provide a foundation for the rational design of future fluorescent probes with improved performance.
The experimental methodologies established during the fellowship, including synthesis of model fluorophore systems and comprehensive ultrafast spectroscopic characterization, constitute useful tools that can be applied to future studies of molecular photophysics, fluorescence sensing, and energy-transfer processes.
Further uptake of the project results will primarily require additional fundamental research. In particular, clarification of the solvent-dependent FRET mechanism, extension of the studies to more complex molecular architectures, and validation in biologically relevant environments would strengthen the scientific significance of the findings. The project is expected to result in peer-reviewed publications, thereby disseminating the developed methodologies and mechanistic insights to the broader scientific community. At this stage, no intellectual property protection, commercial exploitation, or standardization activities are envisaged.