Challenges In Sample Preparation Research Articles

High-fidelity Image Restoration of Large 3D Electron Microscopy Volume.

Volume electron microscopy (VEM) is an essential tool for studying biological structures. Due to the challenges of sample preparation and continuous volumetric imaging, image artifacts are almost inevitable. Such image artifacts complicate further processing both for automated computer vision methods and human experts. Unfortunately, the widely used contrast limited adaptive histogram equalization (CLAHE) can alter the essential relative contrast information about some biological structures. We developed an image-processing pipeline to remove the artifacts and enhance the images without CLAHE. We apply our method to VEM datasets of a Microwasp head. We demonstrate that our method restores the images with high fidelity while preserving the original relative contrast. This pipeline is adaptable to other VEM datasets.

Affordable and Reliable RP-HPLC Method for Verapamil Hydrochloride Quantification in Rabbit Plasma for Pharmacokinetics

Background: Existing bioanalytical methods for verapamil hydrochloride (VH) are often complex, requiring advanced instrumentation and specialized expertise, which limits their use in resource-constrained laboratories. Aim: The goal of this study is to fill this gap by developing a simplified, robust RP-HPLC-UV approach for the estimation of verapamil hydrochloride in rabbit plasma. Designed to enhance accuracy and precision while minimizing sample preparation challenges, this method addresses existing limitations by providing an affordable and reliable alternative for laboratories lacking sophisticated instrumentation. Methods: The bioanalytical method was implemented on C-18 stationary phase (5 μ, 250 × 4.6 mm) using acetonitrile/0.1% tetrahydrofuran (THF) in water (80:20, in volume) as the liquid system at a 1 mL/min flow speed, employing carvedilol as an internal standard. Results: The reported retention times of verapamil hydrochloride and carvedilol were ~7.64 and 4.69 min, respectively, at sufficiently high system suitability standards. The linearity of the bioanalytical approach can be seen between 0.025 and 5.0 µg/mL (r2 = 0.9991). The findings indicated that there was no matrix influence in terms of accuracy (≥98.96 ± 2.68%), intra- and inter-day precision (≤3.68%), recovery (101.98 ± 2.76%), and procedure efficiency (100.65 ± 1.82%). Benchtop, long-term, and short-term stability investigations all revealed that the verapamil hydrochloride in the bio-samples was stable. The pharmacokinetic parameters (Cmax—3.47 µg/mL; Tmax—1.59 h) were studied from time-dependent plasma concentrations of verapamil hydrochloride estimated after 40 mg oral dosing in New Zealand white rabbits. Conclusions: The developed bioanalytical method provided easier quantitative analysis of verapamil hydrochloride from rabbit plasma and was effectively used in a pharmacokinetic investigation of an oral bolus. The reliable performance of this method under practical conditions positions it as a crucial tool for advancing pharmacokinetic studies across various research environments.

From bottom-up to cell surface proteomics: detergents or no detergents, that is the question.

Measuring the expression levels of membrane proteins (MPs) is crucial for understanding cell differentiation and tissue specificity, defining disease characteristics, identifying biomarkers, and developing therapeutics. While bottom-up proteomics addresses the need for accurately surveying the membrane proteome, the lower abundance and hydrophobic nature of MPs pose challenges in sample preparation. As MPs normally reside in the lipid bilayer, conventional extraction methods rely on detergents, introducing here a paradox - detergents prevent aggregation and facilitate protein processing, but themselves become contaminants that interfere with downstream analytical applications. Various detergent removal methods exist to mitigate this issue, including filter-aided sample preparation, SP3, suspension trapping, and membrane mimetics. This review delves into the fundamentals of each strategy, applications, merits, and limitations, providing insights into their effectiveness in MP research.

Rapid Freezing and Cryo-SEM-EDS Imaging of Foraminifera (Unicellular Eukaryotes) Using a Conductive Viscous Cryogenic Glue.

Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.

Correlative single-cell hard X-ray computed tomography and X-ray fluorescence imaging

X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.

Imaging actin organisation and dynamics in 3D.

The actin cytoskeleton plays a critical role in cell architecture and the control of fundamental processes including cell division, migration and survival. The dynamics and organisation of F-actin have been widely studied in a breadth of cell types on classical two-dimensional (2D) surfaces. Recent advances in optical microscopy have enabled interrogation of these cytoskeletal networks in cells within three-dimensional (3D) scaffolds, tissues and in vivo. Emerging studies indicate that the dimensionality experienced by cells has a profound impact on the structure and function of the cytoskeleton, with cells in 3D environments exhibiting cytoskeletal arrangements that differ to cells in 2D environments. However, the addition of a third (and fourth, with time) dimension leads to challenges in sample preparation, imaging and analysis, necessitating additional considerations to achieve the required signal-to-noise ratio and spatial and temporal resolution. Here, we summarise the current tools for imaging actin in a 3D context and highlight examples of the importance of this in understanding cytoskeletal biology and the challenges and opportunities in this domain.

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

Single-Nucleus RNA-Sequencing in Brain Tissue.

Neuroscience research greatly benefits from single-cell sequencing technologies, which can reveal transcriptional alterations on a cellular level. However, preparing single-cell suspensions is technically challenging, requires experience, and has several limitations that can influence the transcriptional readout. Performing sequencing of single nuclei instead of single cells alleviates several of the challenges of sample preparation and highlights acute nuclear transcription. Here, we provide a protocol to prepare a nuclei suspension for single-nucleus RNA-sequencing for cell type-specific transcriptional profiling of brain tissue using the 10x Genomics single-cell gene expression assay. Furthermore, we highlight important aspects to consider during experimental design and data analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of single-nucleus suspension Basic Protocol 2: Preparation and sequencing of single-nucleus libraries for RNA-seq.

3DCNAS: A universal method for predicting the location of fluorescent organelles in living cells in three-dimensional space

Cellular biology research relies on microscopic imaging techniques for studying the complex structures and dynamic processes within cells. Fluorescence microscopy provides high sensitivity and subcellular resolution but has limitations such as photobleaching and sample preparation challenges. Transmission light microscopy offers a label-free alternative but lacks contrast for detailed interpretation. Deep learning methods have shown promise in analyzing cell images and extracting meaningful information. However, accurately learning and simulating diverse subcellular structures remain challenging. In this study, we propose a method named three-dimensional cell neural architecture search (3DCNAS) to predict subcellular structures of fluorescence using unlabeled transmitted light microscope images. By leveraging the automated search capability of differentiable neural architecture search (NAS), our method partially mitigates the issues of overfitting and underfitting caused by the distinct details of various subcellular structures. Furthermore, we apply our method to analyze cell dynamics in genome-edited human induced pluripotent stem cells during mitotic events. This allows us to study the functional roles of organelles and their involvement in cellular processes, contributing to a comprehensive understanding of cell biology and offering insights into disease pathogenesis.

Challenges in sample preparation for swellable core technology tablets: Approaches for the removal of polyethylene oxide and optimization of API recovery

Swellable Core Technology (SCT) tablets, a solid oral dosage formulation designed for the controlled release of Active Pharmaceutical Ingredient (API), are made up of two distinct layers; an active layer containing the active ingredient (10–30%wt) and up to 90%wt polyethylene oxide (PEO); and a sweller layer which contains up to 65%wt PEO. The objective of this study was to develop a process to remove PEO from analytical test solutions and optimize API recovery using physicochemical properties of the API. Quantitation of PEO was performed by liquid chromatography (LC) using an evaporative light scattering detector (ELSD). This was used to build an understanding of removal of PEO using solid-phase extraction and liquid-liquid extraction techniques. A workflow was proposed to allow efficient development of analytical methods for SCT tablets with optimized sample clean-up.

Making Connections: Challenges and Opportunities for In Situ TEM Biasing

Abstract This article discusses sample preparation challenges that have impeded progress in producing bias-enabled TEM samples from electronic components, as well as strategies to mitigate these issues.

New challenges in sample preparation: Miniaturized stir bar sorptive dispersive microextraction as a high-throughput and feasible approach for low-availability sample preparation

The miniaturization of stir bar sorptive dispersive microextraction (mSBSDME) for the analysis of low-availability samples is presented. This new methodology is based on the principles of stir bar sorptive dispersive microextraction, but the amount of sorbent and, most importantly, the amount of sample are considerably reduced to a tiny amount and a few microliters, respectively. Thus, affordable 400-μL flat-base glass inserts and minute bar-shape neodymium magnets (3 mm length x 2 mm diameter) were used as extraction devices hold by a specifically designed multiextraction assembly, which comprises a high-rate stirring plate and a 3D-printed support to treat 15 samples simultaneously. This new approach allows a fast, affordable, portable, and high-throughput analysis of low-volume samples, expanding the potential of the technique. The same extraction device is used along the different stages, thus avoiding transfers, which reduces sample handling. Besides, the reduction in the sample, sorbent and organic solvent amounts allows a considerable decrease of the waste generation, and thus pursues a green sample preparation for bioanalysis. As a proof-of-concept of this new methodology, cortisone and cortisol were determined in human saliva using a composite material made of a reversed phase polymer (Strata™-X-RP) and CoFe2O4 magnetic nanoparticles. Liquid chromatography coupled to tandem mass spectrometry was used to measure both analytes obtaining good analytical features in terms of linearity (R2 > 0.997), method limits of detection and quantification (22.6 and 75.5 ng L−1 for cortisone, and 19.3 and 64.3 ng L−1 for cortisol, respectively), repeatability (RSD ≤11%) and relative recoveries (78–134%).

Advancement and challenges in sample preparation for atomic force microscopy and infrared microscopy for wood-based materials.

Wood-based materials such as composites or laminates play an important role in today's furniture industry, especially in manufacturing high-quality kitchen and dining room furniture. One important aspect after fabrication is the investigation of these materials to derive quality metrics such as surface stain and scuff resistance. Current sample preparation methods are mostly straightforward and rely on cutting and grinding the materials under test, including sensitive wood substrates. To investigate cross-sections and different layer topologies, characterisation techniques such as atomic force microscopy (AFM) and infrared (IR) microscopy are of potential interest. However, a huge limitation is that current sample preparation methods lead to smeared coatings on the sample cross-sections and high surface roughness. Hence, these methods are not applicable for the sample preparation in measuring AFM and IR microscopy. Therefore, new preparation techniques need to be developed accordingly. This article presents a new approach towards coated wood-based sample preparation including embedding processes to use those samples for AFM and IR microscopy technologies. The proposed method has been evaluated by obtaining AFM, IR and microscopy measurements of more than four different wood-based samples such as (i) raw paper, (ii) impregnated paper, (iii) melamine-coated chipboards and (iv) medium-density fibreboards. The investigation results showed a significant improvement in sample preparation, as well as clear chemical and physical characterisation over whole sample construction, including coating layers, for wood-based materials.

High-throughput tensile testing of silver nanowires

Silver nanowires (Ag NWs) have been used in various nanostructured materials such as stretchable electronics due to their small size, high strength, and high conductivity. Envisioned nanostructured materials employ millions of nanostructures to achieve its function, yet due to challenges in sample preparation, mechanical characterization has been limited to a small number of samples. The results thus have limited statistical significance, important, for example, for the case of fracture strain, which is a stochastic variable. Here, we show highly-dense and unidirectional alignment of penta-twinned Ag NWs on top of polyethylene terephthalate (PET) substrates using microfluidics. Surface treatment enabled strong bonding between NWs and substrates. We then conducted tensile stretching of the substrates in the direction of alignment, capturing images of the induced deformation and fracture of the NWs, effectively testing many of them in tension simultaneously. We analyzed their fracture strain using digital image correlation (DIC). We show results for more than 200 samples, achieving a statistical quantification of NW fracture strain, useful for the design of applications and nanostructured materials based on them, such as stretchable electronics.

Rapid solidification of hypoeutectic aluminum copper alloys using fast-scanning calorimetry

The rapid solidification behaviour of binary aluminum copper alloys in the hypoeutectic range of compositions has been studied in this work. Solidification at cooling rates up to 40,000 °Cs−1 can be achieved using a chip-based calorimeter, but present challenges in sample preparation and temperature correction, which are addressed in this work. Data from calorimetry has been used to produce fraction of solid curves which are important input to metal manufacturing processing models. Undercoolings range from around 50 °C at 100 °Cs−1 to around 100 °C at 40,000 °Cs−1. From these, a kinetic phase diagram was constructed for the hypoeutectic region of the Al-Cu system, for cooling rates up to 40,000 °Cs−1. The undercoolings measured in this experiment are considerably higher than those predicted theoretically for growth-based rapid solidification, but not as high as those predicted under homogeneous nucleation.

Nanoscale Compositional Mapping of Commercial LiNi0.8Co0.15Al0.05O2 Cathodes Using Atom Probe Tomography

Nickel-rich cathodes provide improved specific capacity, which leads to higher gravimetric energy density, which, in turn, is critical for electric vehicles. However, poor long-term capacity retention at elevated temperatures/high C rates (the rate of charge and discharge on a battery) stems from material issues: surface phase changes, corrosive side reactions with the electrolyte, ion dissolution, and propagation of cracks. Introducing dopants, developing nanoscale surface coatings, and graded core–shell structures all improved the electrochemical performance of nickel-rich cathodes. However, material-level understanding of the effect of Li composition and distribution in Ni-rich cathodes is limited due to a lack of characterization methods available that can directly image Li at the nanoscale. Hence, it is critical to establish methods such as atom probe tomography (APT) that have both nanometer-scale spatial resolution and high compositional sensitivity to quantitatively analyze battery cathodes. To fully realize its potential as a method for quantitative compositional analysis of commercial Li-ion batteries, we provide a comprehensive description of the challenges in sample preparation and analyze the dependency of the analysis parameters, specifically laser pulse energy on the measured stoichiometry of elements in a high-Ni-content cathode material LiNi0.8Co0.15Al0.05O2 (NCA). Our findings show that the stoichiometry variations cannot be explained by charge–state ratios or Ga implantation damage alone during FIB preparation, indicating that additional factors such as crystallographic orientation may need to be considered to achieve quantitative nanoscale compositional analysis of such battery cathodes using APT.

Measuring H2O concentrations in olivine by secondary ion mass spectrometry: Challenges and paths forward

Abstract Trace concentrations of H2O in olivine strongly affect diverse mantle and magmatic processes. H2O in olivine has been difficult to accurately quantify due to challenges in sample preparation and measurement, as well as significant uncertainties in standard calibrations. Here we directly compare secondary-ion mass spectrometry (SIMS) measurements of the olivine standards of Bell et al. (2003, hereafter Bell03) and Withers et al. (2012, hereafter Withers12) upon which most SIMS and Fourier transform infrared (FTIR) spectroscopy analyses are based. In the same SIMS session, we find that the olivine standards from the two studies are offset by ~50%, forming lines of different slope when comparing SIMS measurements to the independent nuclear reaction analysis (NRA) in Bell03 and elastic recoil detection analysis (ERDA) in Withers12. This offset is similar to the ~40% offset that exists in the FTIR absorption coefficients determined by those two studies, and points to the NRAERDA data as the cause for the offset more than different IR absorption characteristics of the different olivines. We find that the Withers12 olivine standards form the most precise calibration line, and that the measured Bell03 olivine standards have issues of reproducibility and accuracy due to the presence of hydrous inclusions (as documented previously by Mosenfelder et al. 2011). Owing to the limited availability of the Withers12 olivine standards, however, we recommend using orthopyroxene standards (Kumamoto et al. 2017) to calibrate H2O in olivine by SIMS due to similar calibration slopes. We revise the reference values of current orthopyroxene standards to account for uncertainties in the Bell et al. (1995) manometry data. With these revised values, the orthopyroxene calibration line is within 12% of the Withers12 olivine line, which is within the long-term uncertainty of the SIMS olivine measurements. We apply our SIMS calibration protocol to revise estimates of the partition coefficients for H2O between olivine and melt, resulting in a value of 0.0009 ± 0.0003 at pressures ~0.2–2 GPa. This brings into closer agreement between the partition coefficients determined from experimental studies and those based on natural studies of olivine-hosted melt inclusions.

ICP-MS-based Approach to Determine Nanoparticle Recovery After Hollow Fiber Flow Field Flow Fractionation.

Compared to the classical chemicals, nanoparticles (NPs) exhibit unique properties, which lead to challenges in sample preparation and analysis. Fractionation techniques and, in particular, hollow fiber flow field flow fractionation (HF5) have recently become popular in the characterization and quantification of nanomaterials, because of their fine fractionation capability in the nanoscale-range. When dealing with NPs, a great drawback during fractionation is the loss of particles in the fractionation devices, tubing and connectors. There is a need for studies to systematically explore and assess the quality of the fractionation process. A combination of two complementary mass-based setups was used to determine particle loss in HF5. Inductively coupled plasma mass spectrometry (ICP-MS) enabled the estimation of recovery rates for NPs after HF5 separation. Reciprocally, laser ablation ICP-MS (LA-ICP-MS) permitted the evaluation of particles retained on the hollow fiber. 15 nm Au-NPs in different concentrations were evaluated in this study and showed a recovery level for Au-NPs of 50 - 65% based on the applied concentrations after a complete HF5 separation run. Detection of sample deposition on the hollow fiber by LA-ICP-MS indicated a sample loss of about 8%. These findings are important for experiments relying on fractionation of low concentrated nanoparticulate samples.

Agile fusion method for the determination of Pu isotopes in diverse sediments

Atmospheric nuclear testing has occurred worldwide resulting in trace amounts of plutonium (Pu) isotopes in soils and sediments across the globe. Soils and sediments can vary greatly in both physical and chemical properties within close geographic regions presenting a major challenge for sample preparation in the analysis of fallout radionuclides. It is vital to have a rapid, agile method for the complete digestion of samples containing refractory Pu particles to address this challenge. A radioanalytical procedure for the analysis of Pu isotopes in soils/sediments has been developed improving sample preparation using fusion. This is done by eliminating the need for pre-dissolution combustion to remove organic material and providing greater application to a diverse range of samples including those of metal-bearing mineralogy. The integration of borate and peroxide flux materials creates a sample dissolution procedure that is greater than the sum of its parts. This method is applicable to the analysis of 239/240Pu in soil/sediment samples with a sample mass of 3 g, ranging from 0.05 to 5000 mBq/g achieving 242Pu tracer recoveries between 50 and 94%, with a trace-level detection limit of 0.04 mBq/g and measurement uncertainties down to 19%; adjustable to measurement priorities with variation in count time, background counts, chemical recovery and total activity of the sample. This sample preparation technique can be utilized with complementary analysis of alpha spectrometry or ICP-MS. Achieving these parameters provides a highly adaptable standard method for 239/240Pu in soil/sediment analysis applicable to samples from radioecological research at trace environmental levels to emergency response scenarios with 48-h turn-around producing highly repeatable, succinct and high-quality analysis.

Recent Advances in EBSD Characterization of Metals

Electron backscatter diffraction (EBSD) has been attracting enormous interest in the microstructural characterization of metals in recent years. This characterization technique has several advantages over conventional ones, since it allows obtaining a wide range of characterization possibilities in a single method, which is not possible in others. The grain size, crystallographic orientation, texture, and grain boundary character distribution can be obtained by EBSD analysis. Despite the limited resolution of this technique (20–50 nm), EBSD is powerful, even for nanostructured materials. Through this technique, the microstructure can be characterized at different scales and levels with a high number of microstructural characteristics. It is known that the mechanical properties are strongly related to several microstructural aspects such as the size, shape, and distribution of grains, the presence of texture, grain boundaries character, and also the grain boundary plane distribution. In this context, this work aims to describe and discuss the possibilities of microstructural characterization, recent advances, the challenges in sample preparation, and the application of the EBSD in the characterization of metals.