Bio-relevant anion radicals excited states dynamics study in solution by pump–repump–probe spectroscopy – Rad3P

The RAD3P project combines several advanced technologies to implement a unique three-pulse “Pump (electrons) – Repump (femtosecond laser) – Probe (supercontinuum)” spectroscopy on the ELYSE platform. The first component is picosecond electron pulse radiolysis, which generates presolvated and solvated electrons and produces the transient anion radicals that serve as the initial species for excitation. This provides a direct entry point into the primary steps of radiation chemistry.

Selective excitation of these radicals is achieved using a tunable femtosecond optical parametric amplifier (OPA), which enables access to UV–VIS–IR wavelengths, including the critical 330–350 nm region associated with nucleotides. The ultrashort duration and tunability of the OPA pulses enable promotion of anion radicals to excited electronic states and the exploration of their relaxation dynamics.

Time-resolved detection is performed with a broadband supercontinuum probe covering UV to near-IR. This probe enables the measurement of transient absorption signals with femtosecond resolution, allowing the monitoring of bleaching, excited-state absorption, and recombination processes over a wide spectral window.

Precise temporal control between the three pulses is achieved using high-precision optical delay lines that can scan femtosecond-to-nanosecond delays. Spatial overlap is maintained through a motorized alignment and beam-tracking system, based on CCD cameras and remotely controlled mirrors, ensuring stable conditions during long scans.

The experiment is supported by diagnostic tools (energy meters, spectral monitoring, CCD imaging) and by a modern Python-based control framework using PyTango and Taurus. This software infrastructure replaces legacy LabVIEW systems, synchronizes instrument control and data acquisition, and automatically records metadata. A machine-learning algorithm was developed to filter transient signals based on signal-to-noise quality during acquisition.

Together, these technologies enable, for the first time, a fully integrated three-pulse ultrafast radiolysis experiment capable of probing the excited-state dynamics of anion radicals in solution.

Although the full scientific program could not be completed due to prolonged technical limitations, the RAD3P project achieved several important advancements in instrumentation, methodology, and preliminary ultrafast measurements. These results significantly strengthen the ELYSE platform and make the three-pulse technique feasible for future studies.

A complete reconstruction of the optical and mechanical layout was carried out. This included redesigning beam paths, integrating new delay lines, installing motorized mirror systems, and improving optical stability around the accelerator direct line. The installation of the TOPAS OPA and its wavelength-mixing unit enabled tunable UV–VIS–IR excitation, enabling UV transient absorption measurements for the first time on ELYSE (e.g., CO₂•– under pressure). Although the OPA suffered several failures during the project, its partial operation demonstrated that tunable femtosecond excitation is achievable.

A beam-tracking and overlap system, based on CCD cameras and motorized mirrors, was implemented to stabilize alignment between electron, pump, and probe beams. White-light supercontinuum generation was improved, and new optical delay lines allowed controlled femtosecond–nanosecond time delays. Synchronization between electron pulses and the femtosecond laser, initially unstable by several picoseconds, was progressively restored and became reliable at the end of the project. Planned upgrades in 2026 (new laser head and synchronization electronics) will complete this stabilization.

The experiment's control architecture was modernized using Python-based software (PyTango, Taurus), replacing legacy LabVIEW systems. Metadata handling, automatic device control, and a machine-learning algorithm for SNR-based signal selection were also developed, providing a robust foundation for future high-throughput measurements.

On the scientific side, the first successful pump–repump–probe measurements of the solvated electron in water were obtained. The transient bleaching and recovery dynamics showed lifetimes around 500 fs, in agreement with the current non-adiabatic solvation model. Test experiments in concentrated salt solutions and in ethanol confirmed the method's feasibility, although systematic studies require full restoration of OPA and IR detection. THF measurements could not be completed due to a detector malfunction.

While experiments on nucleotides, nucleosides, bases, and peptides could not be carried out within the project timeframe, the technical groundwork is now largely completed. The three-pulse setup is close to operational, and the remaining hardware upgrades will allow the scientific objectives of RAD3P to be pursued under optimal conditions.

The outstanding feature of RAD3P is the creation of a new experimental capability that does not currently exist anywhere else: a three-pulse ultrafast radiolysis technique combining picosecond electron pulses, femtosecond tunable excitation, and broadband supercontinuum probing. This approach enables access to the excited-state dynamics of anion radicals in solution on femtosecond timescales, a domain that has remained experimentally inaccessible until now. The ability to generate anions by electron attachment and then excite them within the same experimental sequence represents a major conceptual and methodological innovation.

Although significant technical challenges delayed its full implementation, the project has brought the ELYSE platform very close to operating this technique under stable and reproducible conditions. Key components — optical layout, beam-tracking, delay lines, synchronization electronics, software infrastructure, and advanced diagnostics — have been developed or modernized. The project, therefore, leaves behind a substantially upgraded instrument with improved stability, better control, larger spectral capabilities, and stronger automation.

Prospects are extremely promising. The planned upgrades for 2026 — replacement of the fiber-laser head, installation of new synchronization hardware, complete OPA realignment and crystal replacement, and modernization of motorized controllers — will finalize the setup's stability. Once these upgrades are implemented, the scientific goals originally defined in RAD3P will become fully achievable.

In particular, the technique will allow direct excitation of nucleotide and nucleoside anion radicals in the 330–350 nm range, enabling a unique exploration of their excited states and relaxation pathways. It will also open the way to the first systematic investigation of dissociative electron attachment (DEA) in aqueous solution under controlled and well-defined ultrafast conditions. Extensions to amino-acid and peptide radicals, as well as to systems with high ionic strength or complex solvation environments, will become possible. Beyond biomolecular systems, the technique has potential applications in photophysics, charge-transfer dynamics, and ultrafast solvation studies.

Altogether, RAD3P has laid the technological foundations for a new generation of ultrafast radiation-chemistry experiments. The platform is now positioned to deliver high-impact scientific results once the final hardware improvements are completed.

The objective of the Rad3P project aims to study for the first time the evolution of excited states of transient anion radicals of DNA/peptides models (quantum efficiencies of state-to-state transition) in aqueous solution. Excited-state chemistry of anion radicals is important in the light of dissociative electron attachment (DEA) since it could play role in reductive damage path for DNA/peptides through the formation of the excited anion radicals undergoing irreversible dissociation. In the course of the project Rad3P, unique radiolysis spectroscopy will be developed based on “Pump-Repump-Probe” spectroscopy approach, by replacing Pump with an electron Pulse, what was never performed in radiation studies.

The “Pump(electron pulse)-Repump(laser)-Probe(supercontinuum)” spectroscopy consists of reexciting solute anion radicals formed in reaction with descendants of secondary electrons: presolvated and solvated by a short (100 fs) optical pump (variable energy). The fate of excited anion radicals could be then studied with a proper time resolution with a third spectrally broad 100 fs probe pulse, namely following bleaching of ground-state absorption of the radical anion and the dynamics of excited-state evolution.

The newly generated knowledge will have great importance to radiotherapy techniques fighting cancer, to the codes devoted to the modeling of ionization radiation interaction with matter, DNA/peptide damage which ignores DEA at the moment. In addition, the project addresses both technological and scientific challenges by developing new spectroscopy extending the applicability of pulse radiolysis technique. It will provide a new way for research under ionization radiation since it is clear that there is plenty of room in the short timescale. During this project, we will also implement a data management plan to give access to the community the generated experimental data.