Optimized Sample Preparation Research Articles

Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies.

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19F-NMR.

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and linewidths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed a similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS) for High-Throughput Screening of Pesticides in Rice Samples Collected in Bangladesh and Saudi Arabia

Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS) with modified QuEChERS sample preparation has been applied to the high-throughput screening of pesticide residuals in rice collected from Bangladesh and Saudi Arabia markets. Both countries consume high volumes of rice, which is a fundamental food for their populations. We report optimized sample preparation and mass spectrometry analysis protocols, which can be rapidly deployed in analytical laboratories. The screening of four groups (organophosphorus, synthetic pyrethroid, organonitrogen, and organochlorine) of a total of 115 pesticides can be performed within ~10 min using a matrix-matched calibration. For most compounds, the limits of detection and quantification (LOD/LOQ) are well below the maximum residue levels (MRLs) of the main regulators. The method generally demonstrates acceptable recovery values (91 compounds 75–125% and 10 compounds 30–75%). Out of 55 rice samples analyzed, 16 samples (29%) contained pesticide residues above LOQ. Four samples contained chlorpyrifos with concentrations ranging from 21.3 to 71.9 µg/kg, ten samples contained tebuconazole (34.7–69.0 µg/kg), and three samples contained pirimiphos methyl (10.7–20.7 µg/kg). The concentrations of the pesticide residues detected in these samples are well below MRL of FAO/WHO (chlorpyrifos, 500 µg/kg; tebuconazole, 1500 µg/kg; pirimiphos methyl, 7000 µg/kg).

Large scale screening and quantification of micropollutants in fish from the coastal waters of Cochin, India: Analytical method development and health risk assessment

Urban estuarine and coastal water receive several micropollutants through industrial and agricultural influxes. The bioaccumulation of these micropollutants in fish and their entry into the coastal population's food chain raises significant food safety concerns. Hence, a comprehensive analytical method was developed for ultra-trace level quantification of 345 micropollutants in fish. The optimized sample preparation method could extract compounds suitable for both GC–MS/MS and LC-MS/MS analysis simultaneously. The target list of contaminants included 278 agricultural pesticides and also 102 endocrine disruptors covering polyaromatic hydrocarbons, polychlorinated biphenyls, organochlorines, and endocrine-disrupting pesticides. The GC–MS/MS with large volume injection (LVI) technique, and LC-MS/MS operating in MRM mode, achieved an LOQ of <2.00 ng/g for most of the analytes. The extraction strategy involved tri-phase partitioning between water, acidified acetonitrile, and hexane, followed by salting out. Dispersive solid phase cleanup (dSPE) with C18, Z-Sep+, CaCl2, and MgSO4 was able to reduce the matrix influence, and the method achieved satisfactory recovery in the range of 70.0–120.0 % for all the target analytes. The repeatability and reproducibility relative standard deviation values of the measured analytes were <20.0 %, and the Horwitz ratio values were well below 2. The method was used to accurately measure the target micropollutants in fish from the Cochin estuary, the highly urbanized portion of the Vembanad Lake, and an important Ramsar site. At least one or more of the 41 different micropollutants were identified and quantified in about 90.7 % of the 108 samples analyzed. The importance of large-scale screening and trace-level quantification methods in environmental monitoring and risk assessment is underscored by the results. The risk assessment showed a moderate risk of exposure to the nearby coastal population through the food chain.

Development and validation of multiresidue analysis method for biomonitoring of pesticides and metabolites in human blood and urine by LC-QToF-MS

The widespread use of pesticides and their consequential presence in the environment is a growing concern due to the harmful health effects associated with pesticide exposure. For clinical and toxicology laboratories, a method for simultaneously determining these compounds and their metabolic products in body fluids, such as blood and urine, is important. In the present study, a rapid, sensitive and simultaneous LC-QToF-MS method for detecting multiclass pesticides and metabolites in blood and urine samples has been developed and validated. Four sample preparation procedures, protein precipitation and three different variants of QuEChERS-based extraction were evaluated to find a suitable, simple, and effective sample pretreatment technique. The final optimized sample preparation method (acetonitrile; 400 μl, MgSO4; 40 mg and NaCl; 10 mg) was validated for accuracy, precision, matrix effect, recovery, stability, carryover, and dilution integrity. Analyte recoveries ranged from 75.40 to 113.54 % while accuracy was evaluated in the range of 71.41–108.26 % and precision (%RSD) in the range of 0.01 %–16.85 %. The limit of quantification (LOQ) for all compounds was established in the range of 0.82–7.05 ng mL-1. The developed reliable, robust, and sensitive method was successfully applied for the quantification of target pesticides and metabolites in human blood and urine samples. Evaluated samples resulted in detection of eleven analytes (seven pesticides and four metabolites).

Bullet-DNP Enables NMR Spectroscopy of Pyruvate and Amino Acids at Nanomolar to Low Micromolar Concentrations.

Hyperpolarized pyruvate is a widely used marker to track metabolism in vivo and a benchmark molecule for hyperpolarization methods. Here, we show how a combination of improved bullet-DNP instrumentation, an optimized sample preparation and a further sensitivity increase via a 13C-1H polarization transfer after dissolution enable the observation of pyruvate at a concentration of 250 nM immediately after dissolution. At this concentration, the experiment employs a total mass of pyruvate of only 20 ng or 180 pmol. If the concentration is increased to 45 μM, pyruvate may be detected 1 min after dissolution with a signal-to-noise ratio exceeding 50. The procedure can be extended to observe a mixture of amino acids at low micromolar concentrations.

Optimizing Sample Preparation for Destructive Testing in Resistance Spot Welding

Abstract The integrity and reliability of resistance spot welding, a crucial process in various manufacturing industries, are significantly influenced by the quality of sample preparation. This study focuses on advancing the methodologies and practices in the preparation of samples for resistance spot welding destructive tests, aiming to improve the precision and repeatability of the results. We discuss comprehensive approaches encompassing material selection, surface treatment, dimensional accuracy, and alignment procedures, emphasizing the importance of meticulous handling and preparation techniques, A significant focus is placed on the utilization of sophisticated alignment and measurement tools to enhance the consistency of the welding area subjected to destructive testing. our study demonstrates a significant improvement in the reliability and accuracy of welding machine calibration, ultimately contributing to the overall quality of the welding process. By integrating these refined processes, the study highlights a significant improvement in the reliability of welding tests, contributing to enhanced welding quality and performance in practical applications. Our findings underscore the critical role of detailed and standardized sample preparation in achieving accurate and reliable resistance spot welding destructive test outcomes, providing valuable insights for both industrial applications and further research in welding technology.

(Invited) Aiot-Integrated Digital Imaging Sensor for Molecular-Fingerprint Profiling of Extracellular Vesicles in Liquid Biopsy

The advancement of liquid biopsy techniques for non-invasive tumor analysis holds immense potential in continuously tracking tumor status—recurrence, progression, and treatment response—in real-time. To comprehensively assess tumor evolution, cellular diversity, and drug resistance mechanisms, a pivotal step involves scrutinizing proteins and nucleic acids within extracellular vesicles (EVs) present in biofluids. Our focus lies in crafting an advanced digital bead-based sensor system that seamlessly evaluates the molecular profile of EVs in glioblastoma cases. Our innovative approach enriches tumor-relevant genetic data within EVs using antibody-specific magnetic beads, subsequently amplifying it via reverse transcription recombinase polymerase amplification system across forty thousand droplets. These results are scrutinized using a compact imaging device. The core strengths of our methodology include: (a) Streamlined Sample Processing: Our system facilitates ultra-sensitive miRNA detection in EVs within a rapid ninety-minute window, owing to optimized sample preparation and a compact detection mechanism; (b) Enhanced Signal Clarity: Employing a straightforward isothermal assay significantly boosts the fluorescent signal-to-noise ratio within each 70-µm droplet, achieving over 104 turnovers of fluorescent reporters on a chip; (c) Real-Time Data Analysis: Our system seamlessly captures real-time imaging data for the edge computing, utilizing trained YOLOv5 models for classification, and presents genetic results on the smartphone. Its user-friendly design enables utilization by healthcare workers with varying skill levels. This pioneering approach has the potential to offer affordable, rapid, and informative diagnostics particularly beneficial for underserved regions and healthcare systems facing financial constraints. Furthermore, its adaptability for diverse cancers could revolutionize the pace of diagnosis and treatment decision-making through liquid biopsy, potentially establishing its widespread application in global healthcare.

Matrix effects in ultra-high performance supercritical fluid chromatography-mass spectrometry analysis of vitamin E in plasma: The effect of sample preparation and data processing

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both “bind and elute” and “interferent removal” modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to – 72% for α-tocopherol and from −77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

Combined matrix-assisted laser desorption/ionisation-mass spectrometry imaging with liquid chromatography-tandem mass spectrometry for observing spatial distribution of lipids in whole Caenorhabditis elegans.

Matrix-assisted laser desorption/ionisation-mass spectrometry imaging (MALDI-MSI) is a powerful label-free technique for biomolecule detection (e.g., lipids), within tissue sections across various biological species. However, despite its utility in many applications, the nematode Caenorhabditis elegans is not routinely used in combination with MALDI-MSI. The lack of studies exploring spatial distribution of biomolecules in nematodes is likely due to challenges with sample preparation. This study developed a sample preparation method for whole intact nematodes, evaluated using cryosectioning of nematodes embedded in a 10% gelatine solution to obtain longitudinal cross sections. The slices were then subjected to MALDI-MSI, using a RapifleX Tissuetyper in positive and negative polarities. Samples were also prepared for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using an Exploris 480 coupled to a HPLC Vanquish system to confirm the MALDI-MSI results. An optimised embedding method was developed for longitudinal cross-sectioning of individual worms. To obtain longitudinal cross sections, nematodes were frozen at -80°C so that all worms were rod shaped. Then, the samples were defrosted and transferred to a 10% gelatine matrix in a cryomold; the worms aligned, and the whole cryomold submerged in liquid nitrogen. Using MALDI-MSI, we were able to observe the distribution of lipids within C. elegans, with clear differences in their spatial distribution at a resolution of 5μm. To confirm the lipids from MALDI-MSI, age-matched nematodes were subjected to LC-MS/MS. Here, 520 lipids were identified using LC-MS/MS, indicating overlap with MALDI-MSI data. This optimised sample preparation technique enabled (un)targeted analysis of spatially distributed lipids within individual nematodes. The possibility to detect other biomolecules using this method thus laid the basis for prospective preclinical and toxicological studies on C. elegans.

Optimization of Sample Preparation Procedure for Determination of Fat-Soluble Vitamins in Milk and Infant Food by HPLC Technique

Background: The analysis of vitamins in baby food is a challenging task given the complexity of the food matrix, vitamin stability, and strict regulations of the European Union regarding permissible deviations from declared values. Vitamins in food exist in different concentrations and forms and have different stabilities; thus, the preparation of samples for a reliable analysis using the same procedure is not straightforward. Therefore, significant attention has been devoted to optimizing sample preparation in the analysis of vitamins. Methods: This study aims to determine which of the sample preparation and extraction methods is the most efficient for the simultaneous determination of vitamins A, D, E, and K in milk and baby food using high-performance liquid chromatography (HPLC). Different samples of baby food were prepared in seven different ways based on four methods (saponification, enzymatic hydrolysis, solvent extraction, and solid-phase extraction). Results and Conclusion: According to the validation parameters, the optimal preparation method proved to be solid-phase extraction with a C18 stationary phase, with recoveries of 97.4%, 96.1%, 98.3%, and 96.2% for vitamins A, D, E, and K, respectively, and HPLC with a UV–Vis detector was identified as a sufficiently sensitive technique for the identification and quantification of fat-soluble vitamins in milk and baby food.

Optimization of Green Sample Preparation for the Determination of Hydroxycinnamic Acids in Multi-Floral Honey Using Response Surface Methodology

Hydroxycinnamic acids are an essential phenolic group in honey that are related with its diverse health effects. Furthermore, they can be considered as potential biomarkers for botanical and geographical origin. The extraction of hydroxycinnamic acids from honey requires complicated extraction techniques due to their low contents and matrix particularity. The goal of the present work was to develop a green sample preparation method for the solubilization of hydroxycinnamic acids in honey samples. Thus, a Box–Behnken design has been used to investigate the effect of four factors: (i) percentage of ethanol (60–90%, v/v), (ii) temperature (30–60 °C), (iii) solvent-honey ratio (10–30 mL g−1), and (iv) sonication time (10–40 min) on the contents of caffeic, chlorogenic, and ferulic acids. Based on the desirability index, the ultrasound-assisted mixing of honey with 60.0% (v/v) ethanol at a solvent-honey ratio of 10 mL g−1 for a period of 17.8 min at a temperature of 54.6 °C resulted in the maximum solubilization of hydroxycinnamates. Subsequently, the greenness of developed method was assessed and applied successfully on the analysis of honeys. The present study reports for first time the hydroxycinnamic acid fingerprint of multi-floral honey produced in Cyprus.

Serial block-face scanning electron microscopy of adherent cells on thin plastic substrate

Abstract Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. Here, we present a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. A standard sample preparation and embedding protocol for thin section electron microscopy provided already sufficient resolution and image quality to visualize larger structures. To improve resolution and image quality of SBF imaging, we stepwise tested modifications of the protocol, such as the moderate increase of the heavy metal content of the sample. Modifications of the embedding by either the reduction of the resin layer (minimal embedding) or the addition of silver colloid to the resin were evaluated at high and low vacuum imaging conditions. The optimized sample preparation protocol is very similar to the standard preparation protocol for thin section electron microscopy, so that the samples can also be used for this application. The protocol applies a higher concentration of osmium tetroxide, a higher temperature for heavy metal incubation and an additional lead en bloc staining. In summary, the presented workflow provides a generic and adaptable solution for studying adherent cells by SBF SEM.

SEQUENCING PROTOCOL AND BIOINFORMATICS PIPELINE FOR THE AVIAN INFLUENZA VIRUS

The avian influenza virus (AIV), traditionally confined to avian hosts, has recently been detected in various mammal species, raising significant concerns for both animal and public health, necessitating efficient and accurate methods for virus detection and characterization. This study presents a sequencing protocol combined with a comprehensive bioinformatics pipeline designed for the sequencing and analysis of AIV genomes. The presented streamlined approach encompasses whole genome PCR-amplification of the viral genome, enabling the genome characterization and detection of viral mutations with high precision. An amplicon-based MiniSeq sequencing workflow based on a set of PCR primers targeting all genome segments was developed. Three samples from H5 high pathogenic avian influenza (HPAI) outbreaks in Serbia were sequenced using the MiniSeq platform. The protocol involves optimized sample preparation, tailored specifically for AIV, library preparation and sequencing. This is complemented by a robust bioinformatics pipeline that includes quality control, read mapping, consensus genome generation, subtyping and pathotyping, as well as statistical sequencing data. The pipeline efficiently processes raw sequencing data, ensuring high-quality genome assemblies and accurate identification of viral strains. The protocol was used on AIV samples from various avian species, demonstrating its applicability and reliability. The results highlight the protocol’s capability to generate comprehensive genomic data, which is crucial for monitoring viral evolution and informing public health interventions. The described integrated approach offers a powerful tool for AIV surveillance and research, facilitating timely and informed decision-making in response to avian influenza outbreaks. This protocol can be readily adapted for use in various laboratory settings, contributing to global efforts in combating avian influenza and enhancing our understanding of its genomic landscape.

Robust Quantification of Live-Cell Single-Molecule Tracking Data for Fluorophores with Different Photophysical Properties.

High-speed single-molecule tracking in live cells is becoming an increasingly popular method for quantifying the spatiotemporal behavior of proteins in vivo. The method provides a wealth of quantitative information, but users need to be aware of biases that can skew estimates of molecular mobilities. The range of suitable fluorophores for live-cell single-molecule imaging has grown substantially over the past few years, but it remains unclear to what extent differences in photophysical properties introduce biases. Here, we tested two fluorophores with entirely different photophysical properties, one that photoswitches frequently between bright and dark states (TMR) and one that shows exceptional photostability without photoswitching (JFX650). We used a fusion of the Escherichia coli DNA repair enzyme MutS to the HaloTag and optimized sample preparation and imaging conditions for both types of fluorophore. We then assessed the reliability of two common data analysis algorithms, mean-square displacement (MSD) analysis and Hidden Markov Modeling (HMM), to estimate the diffusion coefficients and fractions of MutS molecules in different states of motion. We introduce a simple approach that removes discrepancies in the data analyses and show that both algorithms yield consistent results, regardless of the fluorophore used. Nevertheless, each dye has its own strengths and weaknesses, with TMR being more suitable for sampling the diffusive behavior of many molecules, while JFX650 enables prolonged observation of only a few molecules per cell. These characterizations and recommendations should help to standardize measurements for increased reproducibility and comparability across studies.

Оптимізація пробопідготовки для визначення 90SR У харчових продуктах

The aim: To determine the possibility of conducting studies of the radionuclide 90Sr in food products in accordance with GN 6.6.1.1-130-2006 «Permissible levels of radionuclides 137Cs and 90Sr in food products and drinking wate»" without concentration by the ashing method. Materials and methods: The article describes the method of calculating the minimum measuring concentration of food products. According to the results of the calculations, the products that can be measured without concentration are determined. Analytical, statistical and computational research methods were used during the work. At the stage of sample preparation for measurement, the question often arises in which form to measure the sample - native or ashed. The specific activities of food products in their native form are in many cases lower than the minimum measurable concentration (MMС) on the spectrometer. Therefore, in order to decide in what form to prepare the sample for research, before starting work, it is necessary to compare the capabilities of the spectrometer, the minimum measured concentration and the permissible level of 90Sr content in the research product. In order to solve the question of the need for ashing of the sample, calculations of MMC were carried out for each food product, as well as the weight of the raw product, necessary to obtain 0.01 kg of ash per measurement was calculated. Results: Tables are compiled based on the results of the calculations. One includes products whose activity can be measured without ashing, and the other includes products with ashing. The use of such tables makes it possible to optimize sample preparation for the study of 90Sr by the spectrometric method. Of the 50 groups of food products, 17 have been identified that can be studied in their native form, which saves time for sample preparation and increases the performance of the spectrometer.

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.

Matrix-assisted laser desorption/ionization matrix incorporation evaluation algorithm for improved peak coverage and signal-to-noise ratio in mass spectrometry imaging.

Despite decades of implementation, the selection of optimal sample preparation conditions for matrix-assisted laser desorption/ionization (MALDI) imaging is still ambiguous due to the lack of a universal and comprehensive evaluation methodology. Thus, numerous experiments with different matrix application conditions accompany a translation of the method to novel sample types and matrices. Mouse brain tissues were covered with 9-aminoacridine through sublimation, followed by recrystallization in vapors of 5% (v/v) methanol solution in water. The samples were analyzed by MALDI time-of-flight mass spectrometry, and the efficiency of lipid and small-molecule ionization was evaluated with different metrics. We first investigate the dependency of matrix density and recrystallization conditions on the thickness of an analyte-empty matrix layer to roughly evaluate the laser shot number required to obtain an intense signal with minimal noise. Then, we introduce metrics for the analysis of small imaging datasets (small sample regions) of model samples based on median quantity of peaks in spectra (medQP) and weighted median signal-to-noise ratio (wmSNR). The evaluation of small regions and taking median values for metrics help overcome the sample heterogeneity and allow for the simultaneous comparison of different acquisition parameters. Here, we propose a methodology based on gradual laser ablation of small regions of sample and further implementation of weighted signal-to-noise ratio to assess various matrix application conditions. The proposed approach helps reduce the number of test samples required to determine optimal sample preparation conditions and improve the overall quality of images.

Optimization of sample preparation, fluorescence- and Raman techniques for environmental microplastics

Microspectroscopic imaging techniques based on spontaneous Raman scattering, Stimulated Raman Scattering (SRS), or fluorescence (with a selective dye) can be used to detect environmental microplastics (MPs) and determine their chemical as well as physical properties. The present study first focuses on optimizing the sample preparation, including a new design for a density separation apparatus and optimization of the Nile Red staining procedure. Tests were carried out with both white and colored reference materials, as well as environmental MPs in a suspended matter sample from the Rhine river. The new ‘MESSY’ system has a mean recovery of 95 ± 5.5 % (three polymer materials, in duplicate). The optimized Nile Red staining allows coarse categorization of MPs into “polar” vs. “non-polar” materials based on their Fluorescence Index (emission wavelength), but fluorescent additives in the polymer can cause misclassification. For unambiguous identification of the polymer type, Raman spectroscopy can be used. Even colored polymers, with or without Nile Red staining, were readily identified by Raman spectroscopy using a red laser (785 nm), except for particles containing carbon black. A Deep-UV Raman microscope (ex = 248.6 nm) was constructed, which allowed identification of all colored plastics, even those pigmented with carbon black. Since unsupervised mapping with spontaneous Raman is very slow, point measurements are preferably used after preselection of particles of interest based on fluorescence imaging. SRS is several orders of magnitude faster than spontaneous Raman mapping but requires multiple scans at different z-heights and at multiple wavenumber settings to detect and identify all particles. The results are expected to contribute to the development of suitable methodologies for the detection and identification of environmental microplastics.