We introduce a novel automated well plate sampling system (AmbiSampler) using laser ablation-rapid evaporative ionization mass spectrometry (LA-REIMS) for label-free high-throughput biochemical screening for small volume biological fluid samples and cell line panels from multiwell plates. The human blood serum samples featured intensive signals in the low m/z range including fatty acids and metabolites and in the higher (m/z > 600) range associated with complex lipids, peptides, and proteins. We demonstrate the current quantification performance of the system through blood serum samples spiked with labelled amino acid mix. Cell line analysis was demonstrated by analyzing the NCI60 human cell lines panel grown in multiwell plates. Without any sample preparation, these experiments yielded rich metabolic and lipidomic mass spectrometry profiles, with classification results showing a 98.7

Hyperspectral imaging is a powerful bioimaging tool which can uncover novel insights, thanks to its sensitivity to the intrinsic properties of materials. However, this enhanced contrast comes at the cost of system complexity, constrained by an inherent trade-off between spatial resolution, spectral resolution, and imaging speed. To overcome this limitation, we present a deep learning-based approach that restores and enhances pixel resolution post-acquisition without any a priori knowledge. Fine-tuned using metrics aligned with the imaging model, our physics-aware method achieves a 16X pixel super-resolution enhancement and a 12X imaging speedup without the need of additional training data for transfer learning. Applied to both synthetic and experimental data from five different sample types, we demonstrate that the model preserves biological integrity, ensuring no features are lost or hallucinated. We also concretely demonstrate the model's ability to reveal disease-associated metabolic changes in Downs syndrome that would otherwise remain undetectable. Furthermore, we provide physical insights into the inner workings of the model, paving the way for future refinements that could potentially surpass instrumental limits in an explainable manner. All methods are available as open-source software on GitHub.

Mass-spectrometry of biological tissue using sub-picosecond laser ablation is investigated experimentally. High relative abundances, including peaks at characteristic m/z, are observed across a range of laser parameters. Neuromorphic imaging is explored as a secondary diagnostic. (c) 2025 The Author(s)

This work demonstrates the combination of ambient laser ablation (LA) with in-source surface-induced declustering, originally developed for rapid evaporative ionization mass spectrometry (REIMS). This combination, termed laser ablation REIMS (LA-REIMS), provides sensitivity, spatial resolution, and chemical coverage comparable to matrix-assisted laser desoprtion ionization (MALDI) but without the requirement for matrix deposition. The atmospheric pressure interface setup was subjected to detailed characterization with regard to geometric and thermal parameters augmented by in-silico flow modeling. The resulting platform was tested using aerosol formed by the infrared laser ablation of tissues. Three different laser systems were successfully employed for ambient mass spectrometric imaging: a carbon dioxide laser (λ = 10.6 μm, τL = ∼100 μs), an optical parametric oscillator (OPO; λ = 2.94 μm, τL = 8 ns), and an optical parametric amplifier (OPA; λ = 3.0 μm, τL = ∼30 ps). Single-cell imaging was achieved using the high-resolving capabilities of the OPA systems, and metabolites and lipids ranging from amino acids through carbohydrates and nuclear bases to complex glycolipids were successfully detected. The technique was also tested as a platform for MS-guided surgery, raising the possibility of using a single technique for generating histological and in vivo data.

Understanding the dynamic cellular metabolism is essential for gaining deeper insights into inter- and intracellular functions. In recent years, mass spectrometry (MS) has become the technology of choice for the biochemical characterization and profiling of cell lines, particularly when coupled with separation techniques such as liquid chromatography (LC-MS). However, these methods typically involve extensive sample preparation with potent organic solvents, which is labor-intensive, time-consuming, and incompatible with direct analysis of intact, live cells. Here, we propose the use of the ambient ionization technique Laser Desorption-Rapid Evaporative Ionization Mass Spectrometry (LD-REIMS) incorporated in an automated platform, for the high-throughput profiling of live or frozen cell monolayers, with minimal pretreatment. Validation experiments using 10 breast and colorectal cancer cell lines confirmed high accuracy, repeatability, and molecular coverage of the method, with over 400 metabolites and lipids detected and identified, including saccharides, amino acids, fatty acids, and glycerophospholipids. Of these, 144 were further confirmed and quantified with LC-MS/MS and standard compounds. We also applied the method to establish lipidomic differences across the isogenic MCF10A cells harboring either WT or MUT PIK3CA. Finally, we conducted time-series experiments on hypoxic cells, which revealed significant dynamic changes in metabolism, including lactate accumulation due to anaerobic glycolysis.

3140 Background: Imprecision in breast-conserving surgery leads to high national average high rates of reoperative intervention. In line with updated margin guidelines, accurate differentiation between non-invasive and invasive breast cancer is essential. This study aimed to assess whether mass spectrometry can distinguish between normal breast tissue, benign, non-invasive and invasive disease towards the development of an intraoperative margin assessment tool. Methods: Breast tissue samples were collected from patients undergoing mastectomy. Samples were flash-frozen, sectioned, and analysed using a Xevo G2-XS QTof mass spectrometer (Waters Corp.). Selected sections were ionised using a pulsed optical parametric oscillator laser (OpoletteTM 2731/3034, OPOTEK) which operated at 2940 nm wavelength and 20 Hz repetition rate. The laser focused on the tissue through a 20 mm focal distance convex lens generating aerosol which was aspirated into the spectrometer. The data was combined using spatial distribution and chemical information from characteristic ions to generate 2D chemical images and labelled using consecutive H&E-stained sections annotated by a Consultant Histopathologist for ground truth cross-validation. Results: Over 1 million mass spectra were collected from imaging 52 breast tissue sections. This includes 720 mass spectra from 31 DCIS breast tissue sections, compared to 6 spectra from 2 DCIS breast tissue samples in previous work. A pixel size of 50 μm and scan rate was 250 μm/s was utilised. An ex-vivo classification model was built using n=6,796 and achieved >99% sensitivity for tumour detection (DCIS and IBC) and 100% specificity for identifying normal tissue. Principal Component Analysis demonstrated accurate separation of IBC, DCIS, benign breast disease, and normal breast tissue. Six possible metabolites were identified following Recursive Feature Elimination (RFE) was used to identify the most significant features which differentiate the tissue types, these were annotated using the Lipid Maps database (http://www.lipidmaps.org/) (Table 1). Cancerous tissue showed higher levels of structural lipids (600-900 Da), while normal/benign breast tissue had higher levels of small metabolites (50-300 Da) and fatty acids (200-400 Da). Conclusions: Mass spectrometry imaging enables accurate differentiation of IBC, DCIS, benign breast disease, and normal breast tissue. Biological features identified from the most significant RFE selected features. m/z value Annotation Delta Theoretical m/z Ion Class 255.2324 Palmitic acid 0.0006 255.2330 M-H Fatty acid 297.2751 FA 19:0 0.0037 297.2799 M-H Fatty acid 307.2019 FA 16:0 0.0026 307.2046 M+Cl Fatty acid 766.5392 PE 38:4 0 766.5392 M-H PE 843.5053 PI 35:4 0.0025 843.5029 M-H PI 891.7444 TG 52:3 0.0003 891.7447 M+Cl TG

We report a 2.94 μm OPA with ~100 ps pulse duration, 400 nJ pulse energy and a 500 kHz repetition rate, which enables high resolution (≤10 μm) laser desorption-based mass spectrometry imaging of biological tissue.

Laser desorption ionization (LDI) is generally considered to be an inferior ionization modality to matrix assisted LDI (MALDI), providing information solely on lipids with low sensitivity. The current study demonstrates that the combination of ambient LDI with in-source surface-induced declustering provides sensitivity and chemical coverage comparable to MALDI. The setup was characterised for infrared laser desorption using two different laser systems and was successfully used for ambient mass spectrometric imaging. 5 µm spatial resolution was achieved enabling single-cell resolution imaging, while metabolites and lipids ranging from amino acids through carbohydrates and nuclear bases to complex glycolipids were successfully detected. The technique was also tested as a platform for MS-guided surgery, raising the possibility of using a single technique for generating histological and in-vivo data. The results suggest that the method can be an important step forward in histological classification for surgery and pathology environments, potentially offering a versatile platform for generating both histological and in vivo data.

Precise manipulation of flexible surgical tools is crucial in minimally invasive surgical procedures, necessitating a miniature and flexible robotic probe that can precisely direct the surgical instruments. In this work, we developed a polymer-based robotic fiber with a thermal actuation mechanism by local heating along the sides of a single fiber. The fiber robot was fabricated by highly scalable fiber drawing technology using common low-cost materials. This low-profile (below 2 millimeters in diameter) robotic fiber exhibits remarkable motion precision (below 50 micrometers) and repeatability. We developed control algorithms coupling the robot with endoscopic instruments, demonstrating high-resolution in situ molecular and morphological tissue mapping. We assess its practicality and safety during in vivo laparoscopic surgery on a porcine model. High-precision motion of the fiber robot delivered endoscopically facilitates the effective use of cellular-level intraoperative tissue identification and ablation technologies, potentially enabling precise removal of cancer in challenging surgical sites.

Tandem mass spectrometry based structural elucidation is currently hampered by our limited understanding of fragmentation chemistry. Here we present the Universal Fragmentation Model (UFM) that is based on gas-phase ion chemistry and modelling and is capable of predicting high-quality fragmentation pathways, structures and energetics for general molecules. We demonstrate that UFM can interpret fragmentation chemistries dominated by complex rearrangements.

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune-generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionization technique for mass spectrometry, laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100% accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas quinolone signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were reduced by more than 75% in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCE This work demonstrates that a high-throughput ambient ionization mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and reduces production of the virulence factors pyocyanin and elastase. No pyocyanin was detectable and elastase levels were reduced by more than 75% in bacteria grown in the presence of hypochlorous acid. This approach has the potential to be widely applicable to the characterization of the stress responses of bacteria.

Treatment for high-grade precancerous cervical lesions and early-stage cancers, mainly affecting women of reproductive age, often involves fertility-sparing treatment methods. Commonly used local treatments for cervical precancers have shown the risk of leaving a positive cancer margin and engendering subsequent complications according to the precision and depth of excision. An intra-operative device that allows the careful excision of the disease while conserving healthy cervical tissue would potentially enhance such treatment. In this study, we developed a polymer-based robotic fiber measuring 150 mm in length and 1.7 mm in diameter, fabricated using a highly scalable fiber drawing technique. This robotic fiber utilizes a hybrid actuation mechanism, combining electrothermal and tendon-driven actuation mechanisms, thus enabling a maximum motion range of 46 mm from its origin with a sub-100 μm motion precision. We also developed control algorithms for the actuation methods of this robotic fiber, including predefined path control and telemanipulation, enabling coarse positioning of the fiber tip to the target area followed by a precise scan. The combination of a surgical laser fiber with the robotic fiber allows for high-precision surgical ablation. Additionally, we conducted experiments using a cervical phantom that demonstrated the robotic fiber's ability to access and perform high-precision scans, highlighting its potential for cervical disease treatments and improvement of oncological outcomes.

By combining high beam quality picosecond pulsed optical parametric amplifiers at 2.94 mu m with Rapid Evaportive Ionisation Mass Spectrometry (REIMS), we have demonstrated record spatial pixel resolutions for ambient mass spectrometry (MS) imaging of < 10 mu m. In this contribution, we introduce our work in this area, demonstrating the platform workflow and highlighting recent results of metabolic imaging at the single cell resolution level.

We report a single-cell level resolution (≤10 µm), laser desorption-based mass spectrometry imaging platform. An optical parametric amplifier is used to generate ∼100 ps, 200 nJ pulses at around 3 µm with a maximum repetition rate of 500 kHz. The pulses are tightly focussed on to fresh frozen animal tissue samples with a thickness of 10 µm. Small volumes of tissue are readily ablated by the laser and are subsequently chemically analyzed using a Rapid Evaporative Ionization Mass Spectrometry (REIMS) source installed on a time of flight mass analyzer. Raster scanning the samples through the laser focus enables the acquisition of mass spectrometry data which can be processed into images with pixel size 10 µm without oversampling, corresponding to cellular level resolution.

Laser desorption ionisation (LDI) is generally considered to be an inferior ionisation modality to matrix assisted LDI (MALDI), providing information solely on lipids with low sensitivity. The current study demonstrates that the combination of ambient LDI with in-source surface-induced declustering provides sensitivity and chemical coverage comparable to MALDI. The setup was characterised for infrared laser desorption using two different laser systems and was successfully used for ambient mass spectrometric imaging. 20 µm spatial resolution was achieved with oversampling, approaching single-cell resolution, while metabolites and lipids ranging from amino acids through carbohydrates and nuclear bases to complex glycolipids were successfully detected. The technique was also tested as a platform for MS-guided surgery, raising the possibility of using a single technique for generating histological and in-vivo data. The results suggest that the new method can form the basis for a new histological classification system for surgery and pathology environments closing this 150 year old diagnostic gap.