Desorption Electrospray Research Articles

High-Throughput Assessment of Bile Salt Export Pump Inhibition Using RapidFire-MS and DESI-MS.

The bile salt export pump (BSEP) assay is widely used to evaluate the potential for drug-induced liver injury (DILI) early in the drug discovery process. While traditional liquid chromatography-mass spectrometry (LC-MS)-based approaches have been utilized for BSEP activity testing, they have intrinsic limitations in either throughput or the requirement for sample preparation and are difficult to scale up in order to screen drug candidates. Here we demonstrate the use of two different high-throughput MS methods based on solid-phase extraction (SPE) and desorption electrospray ionization (DESI) for high-throughput BSEP activity assessment in a label-free manner, with minimal needs for sample workup, at sampling rates of ∼11 and ∼5.5 s/sample, respectively. Both approaches were validated, compared, and successfully applied to the evaluation of 96 drug candidates for the inhibition of taurocholic acid (TCA) transport using BSEP vesicles.

Desorption Electrospray Ionization Cyclic Ion Mobility-Mass Spectrometry Imaging for Traumatic Brain Injury Spatial Metabolomics.

Lipidomics focuses on investigating alterations in a wide variety of lipids that harness important information on metabolic processes and disease pathology. However, the vast structural diversity of lipids and the presence of isobaric and isomeric species creates serious challenges in feature identification, particularly in mass spectrometry imaging experiments that lack front-end separations. Ion mobility has emerged as a potential solution to address some of these challenges and is increasingly being utilized as part of mass spectrometry imaging platforms. Here, we present the results of a pilot mass spectrometry imaging study on rat brains subjected to traumatic brain injury (TBI) to evaluate the depth and quality of the information yielded by desorption electrospray ionization cyclic ion mobility mass spectrometry (DESI cIM MSI). Imaging data were collected with one and six passes through the cIM cell. Increasing the number of passes increased the ion mobility resolving power and the resolution of isobaric lipids, enabling the creation of more specific maps. Interestingly, drift time data enabled the recognition of multiply charged phosphoinositide species in the complex data set generated. These species have not been previously reported in TBI MSI studies and were found to decrease in the hippocampus region following injury. These changes were attributed to increased enzymatic activity after TBI, releasing arachidonic acid that is converted to eicosanoids to control inflammation. A substantial reduction in NAD and alterations in other adenine metabolites were also observed, supporting the hypothesis that energy metabolism in the brain is severely disrupted in TBI.

Spatial metabolomics method to reveal the differences in chemical composition of raw and honey-fried Stemona tuberosa Lour. by using UPLC-Orbitrap Fusion MS and desorption electrospray ionization mass spectrometry imaging.

Stemona tuberosa Lour. (ST) is a significant traditional Chinese medicine (TCM) renowned for its antitussive and insecticidal properties. ST is commonly subjected to processing in clinical practice before being utilized as a medicinal substance. Currently, the customary technique for processing ST is honey-fried. Nevertheless, the specific variations in chemical constituents of ST before and after honey-fried remain unclear. This work aimed to analyze the variations in chemical constituents of ST before and after honey-fried and to study the distribution of differential markers in the roots. UPLC-Orbitrap Fusion MS combined with molecular network analysis was used to analyze the metabolome of ST and honey-fried ST (HST) and to screen the differential metabolites by multivariate statistical analysis. Spatial metabolomics was applied to study the distribution of differential metabolites by desorption electrospray ionization mass spectrometry imaging (DESI-MSI). The ST and HST exhibited notable disparities, with 56 and 61 chemical constituents found from each, respectively. After processing, the types of alkaloids decreased, and 12 differential metabolites were screened from the common compounds. The notable component variations were epibisdehydro-tuberostemonine J, neostenine, tuberostemonine, croomine, neotuberostemonine, and so forth. MSI visualized the spatial distribution of differential metabolites. Our research provided a rapid and effective visualization method for the identification and spatial distribution of metabolites in ST. Compared with the traditional method, this method offered more convincing data supporting the processing mechanism investigations of Stemona tuberosa from a macroscopic perspective.

Protein analysis by desorption electrospray ionization mass spectrometry.

This review presents progress made in the ambient analysis of proteins, in particular by desorption electrospray ionization-mass spectrometry (DESI-MS). Related ambient ionization techniques are discussed in comparison to DESI-MS only to illustrate the larger context of protein analysis by ambient ionization mass spectrometry. The review describes early and current approaches for the analysis of undigested proteins, native proteins, tryptic digests, and indirect protein determination through reporter molecules. Applications to mass spectrometry imaging for protein spatial distributions, the identification of posttranslational modifications, determination of binding stoichiometries, and enzymatic transformations are discussed. The analytical capabilities of other ambient ionization techniques such as LESA and nano-DESI currently exceed those of DESI-MS for in situ surface sampling of intact proteins from tissues. This review shows, however, that despite its many limitations, DESI-MS is making valuable contributions to protein analysis. The challenges in sensitivity, spatial resolution, and mass range are surmountable obstacles and further development and improvements to DESI-MS is justified.

Desorption Electrospray Ionization Mass Spectrometry Imaging of Powder-Treated Fingermarks on Forensic Gelatin Lifters and its Application for Separating Overlapping Fingermarks.

Fingermarks are frequently collected at crime scenes by using gelatin lifters for preservation and transport of the marks to a forensic laboratory for inspection. The gelatin lifters preserve both the imprint of the fingermark pattern necessary for identification purposes and the chemical residue of the mark potentially useful for profiling the person who left the fingermark. The fingermark patterns are traditionally recorded using photography/optical imaging, but methods for chemical analysis of fingermark residues on gelatin lifters are scarce. Here we report the first method for the chemical analysis of fingermarks on gelatin lifters using desorption electrospray ionization mass spectrometry (DESI-MS) imaging. The imaging can be done directly on the gelatin support without any sample preparation, supporting immediate operational use of the method for fingermarks collected at crime scenes. Operational use of the method is further supported by successful chemical imaging of fingermarks enhanced by traditional dusting with forensic powders and lifted off different surfaces (glass, stainless steel, painted aluminum, polystyrene, cardboard, and plastic) as well as fingermarks lifted multiple times. We also demonstrate that the present method can be used to visually separate natural overlapping powder-treated fingermarks, and the chemical composition of the fingermarks can be analyzed on the gelatin support by DESI-MS/MS. The presented method has potential for integration into the traditional workflow for fingermark analysis, and will allow more fingermarks collected at crime scenes to be evaluated both visually and chemically.

Characterisation of Canine and Feline Breast Tumours, Their Metastases, and Corresponding Primary Cell Lines Using LA-REIMS and DESI-MS Imaging.

Breast cancer, a complex disease with a significant prevalence to form metastases, necessitates novel therapeutic strategies to improve treatment outcomes. Here, we present the results of a comparative molecular study of primary breast tumours, their metastases, and the corresponding primary cell lines using Desorption Electrospray Ionisation (DESI) and Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) imaging. Our results show that ambient ionisation mass spectrometry technology is suitable for rapid characterisation of samples, providing a lipid- and metabolite-rich spectrum within seconds. Our study demonstrates that the lipidomic fingerprint of the primary tumour is not significantly distinguishable from that of its metastasis, in parallel with the similarity observed between their respective primary cell lines. While significant differences were observed between tumours and the corresponding cell lines, distinct lipidomic signatures and several phospholipids such as PA(36:2), PE(36:1), and PE(P-38:4)/PE(O-38:5) for LA-REIMS imaging and PE(P-38:4)/PE(O-38:5), PS(36:1), and PI(38:4) for DESI-MSI were identified in both tumours and cells. We show that the tumours' characteristics can be found in the corresponding primary cell lines, offering a promising avenue for assessing tumour responsiveness to therapeutic interventions. A comparative analysis by DESI-MSI and LA-REIMS imaging revealed complementary information, demonstrating the utility of LA-REIMS in the molecular imaging of cancer.

Efficacy of Astragalus membranaceus–Carthamus tinctorius in cerebral ischemia/reperfusion injury: Insights from metabolomics and mass spectrometry imaging

BackgroundThe combination of Astragalus membranaceus and Carthamus tinctorius (AC) exhibits significant therapeutic effects in cerebral ischemia/reperfusion injury (CIRI). Understanding the metabolic characteristics of brain microregions and disturbances in tissues and systemic circulation is crucial for elucidating the mechanisms of CIRI and the therapeutic benefits of AC. However, in situ metabolic regulation of the complex brain structure has not been adequately studied, and the therapeutic mechanism of AC requires immediate clarification. PurposeThe present study aimed to unveil the specific metabolic reprogramming of CIRI at systemic and microregional levels, identify key metabolic pathways and metabolites, and elucidate the therapeutic mechanisms of AC. MethodsAir flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI), a newly developed technique, was used to investigate metabolites in brain microregions. Hematoxylin-eosin, Nissl, and immunofluorescence staining were performed to visualize the microscopic changes associated with spatial metabolism. A comprehensive metabolomics study was conducted on serum, brain tissue, and microregions, along with neurological assessments, cerebral infarction measurements, and Evans blue experiments, to assess the systemic and local metabolic effects of AC treatment for CIRI. ResultsAC significantly reduced neurological damage, minimized infarct size, and repaired blood–brain barrier damage in CIRI rats. AFADESI-MSI demonstrated that the metabolic imbalance caused by CIRI primarily occurs in the cerebral cortex, hippocampus, caudate putamen, thalamus, cerebellar cortex, and fiber tract regions. Significant changes in 16 metabolites were observed in these regions, corresponding to neuron damage, glial cell activation, and neural repair. 20 metabolites from serum and 4 from brain tissue varied significantly with the sham group. Comprehensive metabolomics analysis indicated a close relationship among serum, tissue, and microregional metabolism. CIRI-induced systemic and localized metabolic disorders involve 14 metabolic pathways. AC conferred therapeutic benefits in CIRI by reversing various metabolic imbalances. ConclusionAFADESI-MSI efficiently visualized brain microregion metabolism. Comprehensive metabolomics analysis revealed detailed insights into the specific metabolic reprogramming in CIRI and the therapeutic impacts of AC. AC demonstrated significant clinical potential as an adjunct therapy to existing CIRI treatments.

Unmasking Spatial Heterogeneity in Phytotoxicology Mechanisms Induced by Carbamazepine by Mass Spectrometry Imaging and Multiomics Analyses.

Previous studies have highlighted the toxicity of pharmaceuticals and personal care products (PPCPs) in plants, yet understanding their spatial distribution within plant tissues and specific toxic effects remains limited. This study investigates the spatial-specific toxic effects of carbamazepine (CBZ), a prevalent PPCP, in plants. Utilizing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), CBZ and its transformation products were observed predominantly at the leaf edges, with 2.3-fold higher concentrations than inner regions, which was confirmed by LC-MS. Transcriptomic and metabolic analyses revealed significant differences in gene expression and metabolite levels between the inner and outer leaf regions, emphasizing the spatial location's role in CBZ response. Notably, photosynthesis-related genes were markedly downregulated, and photosynthetic efficiency was reduced at leaf edges. Additionally, elevated oxidative stress at leaf edges was indicated by higher antioxidant enzyme activity, cell membrane impairment, and increased free fatty acids. Given the increased oxidative stress at the leaf margins, the study suggests using in situ Raman spectroscopy for early detection of CBZ-induced damage by monitoring reactive oxygen species levels. These findings provide crucial insights into the spatial toxicological mechanisms of CBZ in plants, forming a basis for future spatial toxicology research of PPCPs.

Oversampling for Enhanced Spatial Resolution of Zebrafish by Top-Hat IR-MALDESI-MSI.

Mass spectrometry imaging (MSI) has become a significant tool for measuring chemical species in biological tissues, where much of the impact of these platforms lies in their capability to report the spatial distribution of analytes for correlation to sample morphology. As a result, enhancement of spatial resolution has become a frontier of innovation in the field, and necessary developments are dependent on the ionization source. More particularly, laser-based imaging sources may require modifications to the optical train or alternative sampling techniques. These challenges are heightened for systems with infrared (IR) lasers, as their operating wavelength generates spot sizes that are inherently larger than their ultraviolet counterparts. Recently, the infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source has shown the utility of a diffractive optical element (DOE) to produce square ablation patterns, termed top-hat IR-MALDESI. If the DOE optic is combined with oversampling methods, smaller ablation volumes can be sampled to render higher spatial resolution imaging experiments. Further, this approach enables reproducible spot sizes and ablation volumes for better comparison between scans. Herein, we investigate the utility of oversampling with top-hat IR-MALDESI to enhance the spatial resolution of measured lipids localized within the head of sectioned zebrafish tissue. Four different spatial resolutions were evaluated for data quality (e.g., mass measurement accuracy, spectral accuracy) and quantity of annotations. Other experimental parameters to consider for high spatial resolution imaging are also discussed. Ultimately, 20 μm spatial resolution was achieved in this work and supports feasibility for use in future IR-MALDESI studies.

Spatial distribution of differential metabolites in different parts of Tetrastigma hemsleyanum Diels et Gilg by ultrahigh-performance liquid chromatography/mass spectrometry and desorption electrospray ionization mass spectrometry imaging

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a homology medicine and food that is rich in bioactive ingredients such as flavonoids and phenolic acids. A strategy that integrates ultrahigh-performance liquid chromatography–electrospray ionization mass spectrometry (UHPLC–ESI/MS) and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) spatial metabolomics analysis was proposed and applied to different parts of T. hemsleyanum for the first time. A total of 132 compounds were tentatively identified, and 3, 7, and 24 components were found only in tuberous roots (TRs), fibrous roots (FRs), and leaves, respectively, using UHPLC–ESI/MS. The DESI-MSI results showed that the spatial distribution of flavonoids between the aerial and underground parts was diverse; for example, vitexin-2″-O-glucoside was largely localized in the epidermis and xylem of TRs, while a small amount was restricted to the xylem of the leaf margin and FRs. There was a higher abundance of most organic acids and flavonoids in the leaves than in the underground parts. Some flavonoids showed the same abundance in FRs and TRs, but the levels of most compounds were considerably higher in FRs. It is possible for leaves and FRs to become new medicinal parts of T. hemsleyanum. Through chemometrics statistics analysis, 19 key differential compounds in different parts of T. hemsleyanum were found, and the pharmacological mechanisms of the main differential metabolites were predicted by network pharmacology. This study provides a novel method for the medicinal development of T. hemsleyanum, showing its great potential in the development of medicinal resources.

Collaborative robotics to enable ultra-high-throughput IR-MALDESI

Over the last 5 years, IR-MALDESI-MS (Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry) has been demonstrated for use in a range of high-throughput biochemical and cellular assays with remarkable sample acquisition rates up to 22 Hz for a single 384-well assay plate. With such high single plate acquisition rates, the rate limiting step becomes how fast subsequent plates can be presented to the MS for analysis. To make this transfer as fast as possible while maintaining safe operation in a laboratory environment, we developed a collaborative robotic plate transfer system (CRPTS) that combines a 6-axis robot with dual plate grippers, a 7th axis conveyor stage, and a 420-plate capacity sample loading window. As a demonstration of the throughput and flexibility of CRPTS, we performed a biochemical assay that monitored the oxidation of tris(2-carboxyethyl)phosphine (TCEP) to screen for nuisance compounds. Using continuous and step motion scan profiles, we analyzed 158,799 compounds contained in 448 assay plates over the course of 12.5 h (Z-Factor=0.87) and 17.5 h (Z-factor=0.99), respectively. Extrapolating these results enables the screening of a million compounds within 6–7 working days.

Revolutionizing food science with mass spectrometry imaging: A comprehensive review of applications and challenges.

Food science encounters increasing complexity and challenges, necessitating more efficient, accurate, and sensitive analytical techniques. Mass spectrometry imaging (MSI) emerges as a revolutionary tool, offering more molecular-level insights. This review delves into MSI's applications and challenges in food science. It introduces MSI principles and instruments such as matrix-assisted laser desorption/ionization, desorption electrospray ionization, secondary ion mass spectrometry, and laser ablation inductively coupled plasma mass spectrometry, highlighting their application in chemical composition analysis, variety identification, authenticity assessment, endogenous substance, exogenous contaminant and residue analysis, quality control, and process monitoring in food processing and food storage. Despite its potential, MSI faces hurdles such as the complexity and cost of instrumentation, complexity in sample preparation, limited analytical capabilities, and lack of standardization of MSI for food samples. While MSI has a wide range of applications in food analysis and can provide more comprehensive and accurate analytical results, challenges persist, demanding further research and solutions. The future development directions include miniaturization of imaging devices, high-resolution and high-speed MSI, multiomics and multimodal data fusion, as well as the application of data analysis and artificial intelligence. These findings and conclusions provide valuable references and insights for the field of food science and offer theoretical and methodological support for further research and practice in food science.

Enhancing drug analysis through advanced mass spectrometry techniques and quantitative modeling

The integration of advanced mass spectrometry (MS) techniques with quantitative modeling has revolutionized the field of drug analysis, offering unprecedented accuracy, sensitivity, and specificity in the detection and quantification of pharmaceutical compounds and their metabolites. High-Resolution Mass Spectrometry (HRMS) and Tandem Mass Spectrometry (MS/MS) have emerged as pivotal tools, capable of distinguishing isobaric species and providing detailed structural information, respectively. Ambient ionization techniques such as Desorption Electrospray Ionization (DESI) and Direct Analysis in Real Time (DART) have further simplified sample preparation, enabling rapid, direct analyses under ambient conditions. This review discusses the significant strides made in enhancing drug analysis through these innovations, alongside the application of calibration curves and mathematical models for quantitation and pharmacokinetic modeling. Additionally, it addresses the challenges inherent in quantitative MS, including matrix effects, analyte stability, and the need for improved sensitivity and specificity. Through a comprehensive overview, this article underscores the transformative impact of these advancements on drug analysis, paving the way for more accurate and efficient pharmaceutical research and diagnostics.

Hardware and software solutions for implementing nanospray desorption electrospray ionization (nano-DESI) sources on commercial mass spectrometers.

Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization mass spectrometry imaging (MSI) approach that enables spatial mapping ofbiological and environmental samples with high spatial resolution and throughput. Because nano-DESI has not yet been commercialized, researchers develop their own sources and interface them with different commercial mass spectrometers. Previously, several protocols focusing on the fabrication of nano-DESI probes have been reported. In this tutorial, we discuss different hardware requirements for coupling the nano-DESI source to commercial mass spectrometers, such as the safety interlock, inlet extension, and contact closure. In addition, we describe the structure of our custom software for controlling the nano-DESI MSI platform and provide detailed instructions for its usage. With this tutorial, interested researchers should be able to implement nano-DESI experiments in their labs.

Integrated spatial metabolomics and network pharmacology to explore the pharmacodynamic substances and mechanism of Radix ginseng-Schisandra chinensis Herb Couple on Alzheimer's disease.

Radix ginseng and Schisandra chinensis have been extensively documented in traditional Chinese medicine (TCM) for their potential efficacy in treating dementia. However, the precise mechanism of their therapeutic effects remains to be fully elucidated. In this study, air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) and network pharmacology are used to investigate the pharmacodynamics and mechanism underlying the herbal combination consisting of Radix ginseng-Schisandra chinensis (RS) in a rodent model for Alzheimer's disease (AD). Brain histopathological findings suggested that RS attenuates hippocampal damage in AD mice, making this combination a potential AD treatment. Twenty-eight biomarkers were identified by spatial metabolomics analysis, which are intricately linked to neuroinflammation, neurotransmitter imbalance, energy deficiency, oxidative stress, and aberrant fatty acid metabolism in AD. The total extract of RS (TE) affected 22 of these biomarkers, with the small molecule components of RS (SN) significantly influencing 19 and the large molecule components of RS (PR) impacting 14. Nine small molecule components are likely to dominate the pharmacodynamics of RS. We constructed a target interaction network based on the corresponding bioactivities that revealed relationships amongst 11 key biomarkers, 8 active ingredients and 12 critical targets. This research illustrates the immense potential of spatial metabolomics and network pharmacology in the study of TCM, revealing the targets and mechanisms underlying herbal formulas.

Tandem mass spectrometry imaging to assess illudin S distributions in Omphalotus illudens mushrooms

Poisoning metabolites within mushrooms typically exert varying physiological and biological effects; however, their distributions within mushrooms are seldom investigated. This study focused on imaging illudin S, a representative poisoning metabolite within Omphalotus illudens mushroom, using desorption electrospray ionization–tandem mass spectrometry (DESI–MS/MS) coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Initially, LC–ESI–MS/MS conditions were established for the selective detection of illudin S (m/z 247→159, positive mode) from extracted mushroom. Based on this MS/MS condition, each mushroom was divided into nine sections, and each section was subjected to specific DESI–MS/MS mode imaging (pixel size: 200 × 200 μm). The results revealed high concentrations of illudin S in the stalks of the mushrooms growing on trees. Further, re-evaluations using the LC–MS/MS assay revealed a seven-fold difference in the illudin S between the stalk and other regions. This study marks the first attempt at assessing toxic metabolite distributions within poisonous mushrooms, thus offering valuable insights including the efficient utilization of illudin analogs in biological applications.

Optimizing neurotransmitter pathway detection by IR-MALDESI-MSI in mouse brain.

Mass spectrometry imaging (MSI) platforms such as infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) are advantageous for a variety of applications, including elucidating the localization of neurotransmitters (NTs) and related molecules with respect to ion abundance across a sample without the need for derivatization or organic matrix application. While IR-MALDESI-MSI conventionally uses a thin exogenous ice matrix to improve signal abundance, it has been previously determined that sucrose embedding without the ice matrix improves detection of lipid species in striatal, coronal mouse brain sections. This work considers components of this workflow to determine the optimal sample preparation and matrix to enhance the detection of NTs and their related metabolites in coronal sections from the striatal region of the mouse brain. The discoveries herein will enable more comprehensive follow-on studies for the investigation of NTs to enrich biological pathways and interpretation related to neurodegenerative diseases and ischemic stroke.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Trans-eyelid distribution of epinastine to the conjunctiva following eyelid application in rabbits

PurposeTo reveal the penetration of epinastine, an anti-allergic ophthalmic agent, into the eyelid and its distribution to the conjunctiva after administration of a cream formulation on rabbit eyelid skin.Study designExperimental study.MethodsRabbits were treated with 0.5% epinastine cream on hair-shaved eyelids, followed by preparation of eyelid tissue slices to determine spatial tissue distribution of epinastine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification using laser-microdissected tissues and desorption electrospray ionization mass spectrometry imaging (DESI-MSI). In addition, following either eyelid application of 0.5% epinastine cream or ocular instillation of 0.1% epinastine eye drops, concentration-time profiles of epinastine in the palpebral conjunctiva and bulbar conjunctiva were determined using LC-MS/MS.ResultsLaser microdissection coupled with LC-MS/MS analysis detected high concentrations of epinastine around the outermost layer of the eyelid at 0.5 h post-administration that gradually diffused deeper into the eyelid and was distributed in the conjunctival layer at 8 and 24 h post-administration. Similar time-dependent drug distribution was observed in high-spatial-resolution images obtained using DESI-MSI. Epinastine concentrations in the conjunctival tissues peaked at 4–8 h after administration of 0.5% epinastine cream and then decreased slowly over 72 h post-administration. In contrast, epinastine concentrations peaked quickly and decreased sharply after epinastine eye drop administration.ConclusionAfter the application of epinastine cream to the eyelid skin, epinastine gradually permeated the eyelid. The compound was retained in the conjunctiva for 8–24 h post-administration, indicating that epinastine cream is a promising long-acting formulation for treating allergic conjunctivitis.

DESI-MSI-guided exploration of metabolic-phenotypic relationships reveals a correlation between PI 38:3 and proliferating cells in clear cell renal cell carcinoma via single-section co-registration of multimodal imaging

A workflow has been evaluated that utilizes a single tissue section to obtain spatially co-registered, molecular, and phenotypical information suitable for AI-enabled image analysis. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to obtain molecular information followed by conventional histological staining and immunolabelling. The impact of varying DESI-MSI conditions (e.g., heated transfer line (HTL) temperature, scan rate, acquisition time) on the detection of small molecules and lipids as well as on tissue integrity crucial for integration into typical clinical pathology workflows was assessed in human kidney. Increasing the heated transfer line temperature from 150 to 450 °C resulted in a 1.8-fold enhancement in lipid signal at a scan rate of 10 scans/s, while preserving histological features. Moreover, increasing the acquisition speed to 30 scans/s yielded superior lipid signal when compared to 10 scans/s at 150 °C. Tissue morphology and protein epitopes remained intact allowing full histological assessment and further multiplex phenotyping by immunofluorescence (mIF) and immunohistochemistry (mIHC) of the same section. The successful integration of the workflow incorporating DESI-MSI, H&E, and immunolabelling on a single tissue section revealed an accumulation of ascorbic acid in regions of focal chronic inflammatory cell infiltrate within non-cancerous kidney tissue. Additionally, a strong positive correlation between PI 38:3 and proliferating cells was observed in clear cell renal cell carcinoma (ccRCC) showing the utility of this approach in uncovering molecular associations in disease pathology.Graphical