The Importance of Spin-Polarized Charge Reorganization in the Catalytic Activity of D-Glucose Oxidase.

The front cover artwork is provided by Prof. Ron Naaman's group at the Weizmann Institute of Science. The image shows that direct electron transfer through GOx is governed by electron spins, which result from the chiral-induced spin selectivity (CISS) effect. Read the full text of the Research Article at 10.1002/cphc.202400033.

Effect of electron beam irradiation pretreatment and different fatty acid types on the formation, structural characteristics and functional properties of starch-lipid complexes.

The effects of different electron beam irradiation doses (2, 4, 8 KGy) and various types of fatty acids (lauric acid, stearic acid, and oleic acid) on the formation, structure, physicochemical properties, and digestibility of starch-lipid complex were investigated. The complexing index of the complexes was higher than 85 %, indicating that the three fatty acids could easily form complexes with starch. With the increase of electron beam irradiation dose, the complexing index increased first and then decreased. The highest complexing index was lauric acid (97.12 %), stearic acid (96.80 %), and oleic acid (97.51 %) at 2 KGy radiation dose, respectively. Moreover, the microstructure, crystal structure, thermal stability, rheological properties, and starch solubility were analyzed. In vitro digestibility tests showed that adding fatty acids could reduce the content of hydrolyzed starch, among which the resistant starch content of the starch-oleic acid complex was the highest (54.26 %). The lower dose of electron beam irradiation could decrease the digestibility of starch and increase the content of resistant starch.

Rapid Structure Determination of Ranitidine Hydrochloride API in Two Crystal Forms Using Microcrystal Electron Diffraction.

The solid-state properties of drug candidates play a crucial role in their selection. Quality control of active pharmaceutical ingredients (APIs) based on their structural information involves ensuring a consistent crystal form and controlling water and residual solvent contents. However, traditional crystallographic techniques have limitations and require high-quality single crystals for structural analysis. Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development. In this study, microED analysis was able to rapidly determine the configuration of two crystal forms (Forms 1, 2) of the API ranitidine hydrochloride. The structures obtained with microED are consistent with previous structures determined by X-ray diffraction, indicating microED is a useful tool for rapidly analyzing molecular structures in drug development and materials science research.

Ternary heterostructure-driven photoinduced electron-hole separation enhanced oxidative stress for triple-negative breast cancer therapy.

Zinc oxide nanoparticles (ZnO NPs) stand as among the most significant metal oxide nanoparticles in trigger the formation of reactive oxygen species (ROS) and induce apoptosis. Nevertheless, the utilization of ZnO NPs has been limited by the shallowness of short-wavelength light and the constrained production of ROS. To overcome these limitations, a strategy involves achieving a red shift towards the near-infrared (NIR) light spectrum, promoting the separation and restraining the recombination of electron-hole (e--h+) pairs. Herein, the hybrid plasmonic system Au@ZnO (AZ) with graphene quantum dots (GQDs) doping (AZG) nano heterostructures is rationally designed for optimal NIR-driven cancer treatment. Significantly, a multifold increase in ROS generation can be achieved through the following creative initiatives: (i) plasmonic Au nanorods expands the photocatalytic capabilities of AZG into the NIR domain, offering a foundation for NIR-induced ROS generation for clinical utilization; (ii) elaborate design of mesoporous core-shell AZ structures facilitates the redistribution of electron-hole pairs; (iii) the incorporation GQDs in mesoporous structure could efficiently restrain the recombination of the e--h+ pairs; (iv) Modification of hyaluronic acid (HA) can enhance CD44 receptor mediated targeted triple-negative breast cancer (TNBC). In addition, the introduced Au NRs present as catalysts for enhancing photothermal therapy (PTT), effectively inducing apoptosis in tumor cells. The resulting HA-modified AZG (AZGH) exhibits efficient hot electron injection and e--h+ separation, affording unparalleled convenience for ROS production and enabling NIR-induced PDT for the cancer treanment. As a result, our well-designed mesoporous core-shell AZGH hybrid as photosensitizers can exhibit excellent PDT efficacy.

First in vitro measurement of VHEE relative biological effectiveness (RBE) in lung and prostate cancer cells using the ARES linac at DESY.

Very high energy electrons (VHEE) are a potential candidate for radiotherapy applications. This includes tumours in inhomogeneous regions such as lung and prostate cancers, due to the insensitivity of VHEE to inhomogeneities. This study explores how electrons in the VHEE range can be used to perform successful in vitro radiobiological studies. The ARES (accelerator research experiment at SINBAD) facility at DESY, Hamburg, Germany was used to deliver 154 MeV electrons to both prostate (PC3) and lung (A549) cancer cells in suspension. Dose was delivered to samples with repeatability and uniformity, quantified with Gafchromic film. Cell survival in response to VHEE was measured using the clonogenic assay to determine the biological effectiveness of VHEE in cancer cells for the first time using this method. Equivalent experiments were performed using 300 kVp X-rays, to enable VHEE irradiated cells to be compared with conventional photons. VHEE irradiated cancer cell survival was fitted to the linear quadratic (LQ) model (R2 = 0.96-0.97). The damage from VHEE and X-ray irradiated cells at doses between 1.41 and 6.33 Gy are comparable, suggesting similar relative biological effectiveness (RBE) between the two modalities. This suggests VHEE is as damaging as photon radiotherapy and therefore could be used to successfully damage cancer cells during radiotherapy. The RBE of VHEE was quantified as the relative doses required for 50% (D0.5) and 10% (D0.1) cell survival. Using these values, VHEE RBE was measured as 0.93 (D0.5) and 0.99 (D0.1) for A549 and 0.74 (D0.5) and 0.93 (D0.1) for PC3 cell lines respectively. For the first time, this study has shown that 154 MeV electrons can be used to effectively kill lung and prostate cancer cells, suggesting that VHEE would be a viable radiotherapy modality. Several studies have shown that VHEE has characteristics that would offer significant improvements over conventional photon radiotherapy for example, electrons are relatively easy to steer and can be used to deliver dose rapidly and with high efficiency. Studies have shown improved dose distribution with VHEE in treatment plans, in comparison to VMAT, indicating that VHEE can offer improved and safer treatment plans with reduced side effects. The biological response of cancer cells to VHEE has not been sufficiently studied as of yet, however this initial study provides some initial insights into cell damage. VHEE offers significant benefits over photon radiotherapy and therefore more studies are required to fully understand the biological effectiveness of VHEE.

In situ electron generation through Fe/C supported sludge coupled with a counter-diffusion biofilm for electron-deficient wastewater treatment: Binding properties and catalytic competition mechanism of nitrate reductase.

A membrane-aerated biofilm-coupled Fe/C supported sludge system (MABR-Fe/C) was constructed to achieve in situ electron production for NO3--N reduction enhancement in different Fe/C loadings (10 g and 200 g). The anoxic environment formed in the MABR-Fe/C promoted a continual Fe2+release of Fe/C in 120 d operation (average Fe2+concentrations is 1.18 and 2.95 mg/L in MABR-Fe/C10 and MABR-Fe/C200, respectively). Metagenomics results suggested that the electrons generated from ongoing Fe2+ oxidation were transferred via the Quinone pool to EC 1.7.5.1 rather than EC 1.9.6.1 to complete the process of NO3--N reduction to NO2--N in Acidovorax, Ottowia, and Polaromonas. In the absence of organic matter, the NO3--N removal in MABR-Fe/C10 and MABR-Fe/C200 increased by 11.99 and 12.52 mg/L, respectively, compared to that in MABR. In the further NO2--N reduction, even if the minimum binding free energy (MBFE) was low, NO2--N in Acidovorax and Dechloromonas preferentially bind the Gln-residues for dissimilatory nitrate reduction (DNR) in the presence of Fe/C. Increasing Fe/C loading (MABR-Fe/C200) caused the formation of different residue binding sites, further enhancing the already dominant DNR. When DNR in MABR-Fe/C200 intensified, the TN in the effluent increased by 3.75 mg/L although the effluent NO3--N concentration was lower than that in MABR-Fe/C10. This study demonstrated a new MABR-Fe/C system for in situ electron generation to enhance biological nitrogen removal and analyzed the NO3--N reduction pathway and metabolic mechanism, thus providing new ideas for nitrogen removal in electron-deficient wastewater.

Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions.

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.

Overlooked Activation Role of Sulfite in Accelerating Hydrated Electron Treatment of Perfluorosulfonates.

Photoexcitation of sulfite (SO32-) is often used to generate hydrated electrons (eaq-) in processes to degrade perfluoroalkyl and polyfluoroalkyl substances (PFASs). Conventional consensus discourages the utilization of SO32- concentrations exceeding 10 mM for effective defluorination. This has hindered our understanding of SO32- chemistry beyond its electron photogeneration properties. In contrast, the radiation-chemical study presented here, directly producing eaq- through water radiolysis, suggests that SO32- plays a previously overlooked activation role in the defluorination. Quantitative 60Co gamma irradiation experiments indicate that the increased SO32- concentration from 0.1 to 1 M enhances the defluorination rate by a remarkable 15-fold, especially for short-chain perfluoroalkyl sulfonate (PFSA). Furthermore, during the treatment of long-chain PFSA (C8F17-SO3-) with a higher concentration of SO32-, the intermediates of C8H17-SO3- and C3F7-COO- were observed, which are absent without SO32-. These observations highlight that a higher concentration of SO32- facilitates both reaction pathways: chain shortening and H/F exchange. Pulse radiolysis measurements show that elevated SO32- concentrations accelerate the bimolecular reaction between eaq- and PFSA by 2 orders of magnitude. 19F NMR measurements and theoretical simulations reveal the noncovalent interactions between SO32- and F atoms, which exceptionally reduce the C-F bond dissociation energy by nearly 40%. As a result, our study offers a more effective strategy for degrading highly persistent PFSA contaminants.

Bio-promoter mediated denitrification recovery from Cd(II) stress: Microbial activity resilience, electron behavior enhancement and microbial community evolution.

Denitrification is fragile to toxic substances, while currently there are few regulation strategies for toxic substance-stressed denitrification. This study proposed a combined bio-promoter composed of basic bio-promoter (cytokinin, biotin, L-cysteine, and flavin adenine dinucleotide) and phosphomolybdic acid (PMo12) to recover cadmium(II) (Cd(II)) stressed denitrification. By inhibiting 58.02% and 48.84% of nitrate reductase and nitrite reductase activities, Cd(II) caused all the influent nitrogen to accumulate as NO3--N and NO2--N. Combined bio-promoter shortened the recovery time by 21 cycles and improved nitrogen removal efficiency by 10% as the synergistic effect of basic bio-promoter and PMo12. Basic bio-promoter enhanced antioxidant enzyme activities for reactive oxygen species clearance and recovered 23.30% of nicotinamide adenine dinucleotide for sufficient electron donors. Meanwhile, PMo12 recovered electron carriers contents, increasing the electron transfer activity by 60.81% compared with self-recovery. Bio-promoters enhanced the abundance of denitrifiers Seminibacterium and Dechloromonas, which was positively correlated with rapid recovery of denitrification performance.

Electronic Properties of DNA Origami Nanostructures Revealed by In Silico Calculations.

DNA origami is a pioneering approach for producing complex 2- or 3-D shapes for use in molecular electronics due to its inherent self-assembly and programmability properties. The electronic properties of DNA origami structures are not yet fully understood, limiting the potential applications. Here, we conduct a theoretical study with a combination of molecular dynamics, first-principles, and charge transmission calculations. We use four separate single strand DNAs, each having 8 bases (4 × G4C4 and 4 × A4T4), to form two different DNA nanostructures, each having two helices bundled together with one crossover. We also generated double-stranded DNAs to compare electronic properties to decipher the effects of crossovers and bundle formations. We demonstrate that density of states and band gap of DNA origami depend on its sequence and structure. The crossover regions could reduce the conductance due to a lack of available states near the HOMO level. Furthermore, we reveal that, despite having the same sequence, the two helices in the DNA origami structure could exhibit different electronic properties, and electrode position can affect the resulting conductance values. Our study provides better understanding of the electronic properties of DNA origamis and enables us to tune these properties for electronic applications such as nanowires, switches, and logic gates.

CERN-based experiments and Monte-Carlo studies on focused dose delivery with very high energy electron (VHEE) beams for radiotherapy applications.

Very High Energy Electron (VHEE) beams are a promising alternative to conventional radiotherapy due to their highly penetrating nature and their applicability as a modality for FLASH (ultra-high dose-rate) radiotherapy. The dose distributions due to VHEE need to be optimised; one option is through the use of quadrupole magnets to focus the beam, reducing the dose to healthy tissue and allowing for targeted dose delivery at conventional or FLASH dose-rates. This paper presents an in depth exploration of the focusing achievable at the current CLEAR (CERN Linear Electron Accelerator for Research) facility, for beam energies >200 MeV. A shorter, more optimal quadrupole setup was also investigated using the TOPAS code in Monte Carlo simulations, with dimensions and beam parameters more appropriate to a clinical situation. This work provides insight into how a focused VHEE radiotherapy beam delivery system might be achieved.

Vibration assisted electron tunnelling in COVID-19 infection using quantum state diffusion.

The spread of the COVID-19 virus has become a global health crisis, and finding effective treatments and preventions is a top priority. The field of quantum biology primarily focuses on energy or charge transfer, with a particular emphasis on photosynthesis. However, there is evidence to suggest that cellular receptors such as olfactory or neural receptors may also use vibration-assisted electron tunnelling to enhance their functions. Quantum tunnelling has also been observed in enzyme activity, which is relevant to the invasion of host cells by the SARS-CoV-2 virus. Additionally, COVID-19 appears to disrupt receptors such as olfactory receptors. These findings suggest that quantum effects could provide new insights into the mechanisms of biological systems and disease, including potential treatments for COVID-19. We have applied the open quantum system approach using Quantum State Diffusion to solve the non-linear stochastic Schrödinger equation (SSE) for COVID-19 virus infection. Our model includes the mechanism when the spike protein of the virus binds with an ACE2 receptor is considered as dimer. These two entities form a system and then coupled with the cell membrane, which is modelled as a set of harmonic oscillators (bath). By simulating the SSE, we find that there is vibration-assisted electron tunnelling happening in certain biological parameters and coupling regimes. Furthermore, our model contributes to the ongoing research to understand the fundamental nature of virus dynamics. It proposes that vibration-assisted electron tunneling could be a molecular phenomenon that augments the lock-and-key process for olfaction. This insight may enhance our understanding of the underlying mechanisms governing virus-receptor interactions and could potentially lead to the development of novel therapeutic strategies.

Electron transfer mediated photo-Fenton-like synergistic catalysis of Fe,Cu-doped MIL-101 coupled with Ag3PO4: Quantitative evaluation and DFT calculations.

Widespread use of tetracycline (TC) results in its persistent residue and bioaccumulation in aquatic environments, posing a high toxicity to non-target organisms. In this study, a bimetal-doped composite material Ag3PO4/MIL-101(Fe,Cu) has been designed for the treatment of TC in aqueous solutions. As the molar ratio of Fe/Cu in composite is 1:1, the obtained material AP/MFe1Cu1 is placed in an aqueous environment under visible light irradiation in the presence of 3 mM peroxydisulfate (PDS), which forms a photo-Fenton-like catalytic system that can completely degrade TC (10 mg/L) within 60 min. Further, the degradation rate constant (0.0668 min-1) is 5.66 and 7.34 times higher than that of AP/MFe and AP/MCu, respectively, demonstrating a significant advantage over single metal-doped catalysts. DFT calculations confirm the strong adsorption capacity and activation advantage of PDS on the composite surface. Therefore, the continuous photogenerated electrons (e-) accelerate the activation of PDS and the production of SO4•-, resulting in the stripping of abundant photogenerated h + for TC oxidation. Meanwhile, the internal circulation of FeⅢ/FeⅡ and CuⅡ/CuⅢ in composite also greatly enhances the photo-Fenton-like catalytic stability. According to the competitive dynamic experiments, SO4•- have the greatest contribution to TC degradation (58.93%), followed by 1O2 (23.80%). The degradation intermediates (products) identified by high-performance liquid chromatography-mass spectrometry (HPLC/MS) technique indicate the involvement of various processes in TC degradation, such as dehydroxylation, deamination, N-demethylation, and ring opening. Furthermore, as the reaction proceeds, the toxicity of the intermediates produced during TC degradation gradually decreases, which can ensure the safety of the aquatic ecosystem. Overall, this work reveals the synergy mechanism of PDS catalysis and photocatalysis, as well as provides technical support for removal of TC-contaminated wastewater.

Plastic scintillator-based dosimeters for ultra-high dose rate (UHDR) electron radiotherapy.

This paper reports the development of dosimeters based on plastic scintillating fibers imaged by a charge-coupled device camera, and their performance evaluation through irradiations with the electron Flash research accelerator located at the Centro Pisano Flash Radiotherapy. The dosimeter prototypes were composed of a piece of plastic scintillating fiber optically coupled to a clear optical fiber which transported the scintillation signal to the readout systems (an imaging system and a photodiode). The following properties were tested: linearity, capability to reconstruct the percentage depth dose curve in solid water and to sample in time the single beam pulse. The stem effect contribution was evaluated with three methods, and a proof-of-concept one-dimensional array was developed and tested for online beam profiling. Results show linearity up to 10 Gy per pulse, and good capability to reconstruct both the timing and spatial profiles of the beam, thus suggesting that plastic scintillating fibers may be good candidates for low-energy electron Flash dosimetry.

Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I.

Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.

Flavonoid Oxidation Potentials and Antioxidant Activities-Theoretical Models Based on Oxidation Mechanisms and Related Changes in Electronic Structure.

Herein, I will review our efforts to develop a comprehensive and robust model for the estimation of the first oxidation potential, Ep1, and antioxidant activity, AA, of flavonoids that would, besides enabling fast and cheap prediction of Ep1 and AA for a flavonoid of interest, help us explain the relationship between Ep1, AA and electronic structure. The model development went forward with enlarging the set of flavonoids and, that way, we had to learn how to deal with the structural peculiarities of some of the 35 flavonoids from the final calibration set, for which the Ep1 measurements were all made in our laboratory. The developed models were simple quadratic models based either on atomic spin densities or differences in the atomic charges of the species involved in any of the three main oxidation mechanisms. The best model takes into account all three mechanisms of oxidation, single electron transfer-proton transfer (SET-PT), sequential proton loss electron transfer (SPLET) and hydrogen atom transfer (HAT), yielding excellent statistics (R2 = 0.970, S.E. = 0.043).

A DFTB study on the electronic response of encapsulated DNA nucleobases onto chiral CNTs as a sequencer.

Sequencing the DNA nucleobases is essential in the diagnosis and treatment of many diseases related to human genes. In this article, the encapsulation of DNA nucleobases with some of the important synthesized chiral (7, 6), (8, 6), and (10, 8) carbon nanotubes were investigated. The structures were modeled by applying density functional theory based on tight binding method (DFTB) by considering semi-empirical basis sets. Encapsulating DNA nucleobases on the inside of CNTs caused changes in the electronic properties of the selected chiral CNTs. The results confirmed that van der Waals (vdW) interactions, π-orbitals interactions, non-bonded electron pairs, and the presence of high electronegative atoms are the key factors for these changes. The result of electronic parameters showed that among the CNTs, CNT (8, 6) is a suitable choice in sequencing guanine (G) and cytosine (C) DNA nucleobases. However, they are not able to sequence adenine (A) and thymine (T). According to the band gap energy engineering approach and absorption energy, the presence of G and C DNA nucleobases decreased the band gap energy of CNTs. Hence selected CNTs suggested as biosensor substrates for sequencing G and C DNA nucleobases.

A high-resolution large-area detector for quality assurance in radiotherapy.

Hadron therapy is an advanced radiation modality for treating cancer, which currently uses protons and carbon ions. Hadrons allow for a highly conformal dose distribution to the tumour, minimising the detrimental side-effects due to radiation received by healthy tissues. Treatment with hadrons requires sub-millimetre spatial resolution and high dosimetric accuracy. This paper discusses the design, fabrication and performance tests of a detector based on Gas Electron Multipliers (GEM) coupled to a matrix of thin-film transistors (TFT), with an active area of 60 × 80 mm2 and 200 ppi resolution. The experimental results show that this novel detector is able to detect low-energy (40 kVp X-rays), high-energy (6 MeV) photons used in conventional radiation therapy and protons and carbon ions of clinical energies used in hadron therapy. The GEM-TFT is a compact, fully scalable, radiation-hard detector that measures secondary electrons produced by the GEMs with sub-millimetre spatial resolution and a linear response for proton currents from 18 pA to 0.7 nA. Correcting known detector defects may aid in future studies on dose uniformity, LET dependence, and different gas mixture evaluation, improving the accuracy of QA in radiotherapy.

4Din vivodosimetry for a FLASH electron beam using radiation-induced acoustic imaging.

Objective. The primary goal of this research is to demonstrate the feasibility of radiation-induced acoustic imaging (RAI) as a volumetric dosimetry tool for ultra-high dose rate FLASH electron radiotherapy (FLASH-RT) in real time. This technology aims to improve patient outcomes by accurate measurements ofin vivodose delivery to target tumor volumes.Approach. The study utilized the FLASH-capable eRT6 LINAC to deliver electron beams under various doses (1.2 Gy pulse-1to 4.95 Gy pulse-1) and instantaneous dose rates (1.55 × 105Gy s-1to 2.75 × 106Gy s-1), for imaging the beam in water and in a rabbit cadaver with RAI. A custom 256-element matrix ultrasound array was employed for real-time, volumetric (4D) imaging of individual pulses. This allowed for the exploration of dose linearity by varying the dose per pulse and analyzing the results through signal processing and image reconstruction in RAI.Main Results. By varying the dose per pulse through changes in source-to-surface distance, a direct correlation was established between the peak-to-peak amplitudes of pressure waves captured by the RAI system and the radiochromic film dose measurements. This correlation demonstrated dose rate linearity, including in the FLASH regime, without any saturation even at an instantaneous dose rate up to 2.75 × 106Gy s-1. Further, the use of the 2D matrix array enabled 4D tracking of FLASH electron beam dose distributions on animal tissue for the first time.Significance. This research successfully shows that 4Din vivodosimetry is feasible during FLASH-RT using a RAI system. It allows for precise spatial (∼mm) and temporal (25 frames s-1) monitoring of individual FLASH beamlets during delivery. This advancement is crucial for the clinical translation of FLASH-RT as enhancing the accuracy of dose delivery to the target volume the safety and efficacy of radiotherapeutic procedures will be improved.