Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefiting from arbitrarily defined pixel sizes. The resolution, speed, sample ...preservation and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of deep learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a 'crappifier' that computationally degrades high SNR, high-pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence time-lapse data, we developed a 'multi-frame' PSSR approach that uses information in adjacent frames to improve model predictions. PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed and sensitivity. All the training data, models and code for PSSR are publicly available at 3DEM.org.
Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of ...COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests.
We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT.
Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation.
Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.
Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify drug residues in wastewater with the aim to estimate illicit drug use at the population level. Transformation ...processes during transport in sewers (chemical and biological reactors) and storage of wastewater samples before analysis are expected to change concentrations of different drugs to varying degrees. Ignoring transformation for drugs with low to medium stability will lead to an unknown degree of systematic under- or overestimation of drug use, which should be avoided. This review aims to summarize the current knowledge related to the stability of commonly investigated drugs and, furthermore, suggest a more effective approach to future experiments. From over 100 WBE studies, around 50 mentioned the importance of stability and 24 included tests in wastewater. Most focused on in-sample stability (i.e., sample preparation, preservation and storage) and some extrapolated to in-sewer stability (i.e., during transport in real sewers). While consistent results were reported for rather stable compounds (e.g., MDMA and methamphetamine), a varying range of stability under different or similar conditions was observed for other compounds (e.g., cocaine, amphetamine and morphine). Wastewater composition can vary considerably over time, and different conditions prevail in different sewer systems. In summary, this indicates that more systematic studies are needed to: i) cover the range of possible conditions in sewers and ii) compare results more objectively. To facilitate the latter, we propose a set of parameters that should be reported for in-sewer stability experiments. Finally, a best practice of sample collection, preservation, and preparation before analysis is suggested in order to minimize transformation during these steps.
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•In-sewer chemical, biological and physical processes influence illicit drug concentrations.•Systematic in-sewer transformation studies covering environmental conditions are needed.•Transformation pathways should be investigated for substances with low or variable stability..•Recommendations for comparable and reliable transformation studies are given.
Lipidomics is a rapidly growing field, fueled by developments in analytical instrumentation and bioinformatics. To date, most researchers and industries have employed their own lipidomics workflows ...without a consensus on best practices. Without a community‐wide consensus on best practices for the prevention of lipid degradation and transformations through sample collection and analysis, it is difficult to assess the quality of lipidomics data and hence trust results. Clinical studies often rely on samples being stored for weeks or months until they are analyzed, but inappropriate sampling techniques, storage temperatures, and analytical protocols can result in the degradation of complex lipids and the generation of oxidized or hydrolyzed metabolite artifacts. While best practices for lipid stability are sample dependent, it is generally recommended that strategies during sample preparation capable of quenching enzymatic activity and preventing oxidation should be considered. In addition, after sample preparation, lipid extracts should be stored in organic solvents with antioxidants at −20 °C or lower in an airtight container without exposure to light or oxygen. This will reduce or eliminate sublimation, and chemically and physically induced molecular transformations such as oxidation, enzymatic transformation, and photon/heat‐induced degradation. This review explores the available literature on lipid stability, with a particular focus on human health and/or clinical lipidomic applications. Specifically, this includes a description of known mechanisms of lipid degradation, strategies, and considerations for lipid storage, as well as current efforts for standardization and quality insurance of protocols.
Abstract
First insights on the human gut microbiome have been gained from medium-sized, cross-sectional studies. However, given the modest portion of explained variance of currently identified ...covariates and the small effect size of gut microbiota modulation strategies, upscaling seems essential for further discovery and characterisation of the multiple influencing factors and their relative contribution. In order to guide future research projects and standardisation efforts, we here review currently applied collection and preservation methods for gut microbiome research. We discuss aspects such as sample quality, applicable omics techniques, user experience and time and cost efficiency. In addition, we evaluate the protocols of a large-scale microbiome cohort initiative, the Flemish Gut Flora Project, to give an idea of perspectives, and pitfalls of large-scale faecal sampling studies. Although cryopreservation can be regarded as the gold standard, freezing protocols generally require more resources due to cold chain management. However, here we show that much can be gained from an optimised transport chain and sample aliquoting before freezing. Other protocols can be useful as long as they preserve the microbial signature of a sample such that relevant conclusions can be drawn regarding the research question, and the obtained data are stable and reproducible over time.
The authors review currently applied collection and preservation methods for gut microbiome research, discussing aspects such as sample quality, applicable omics techniques, user experience and time and cost-efficiency, and evaluate the protocols of the Flemish Gut Flora Project, a large-scale gut microbiome sampling effort in Belgium, to give an idea of perspectives, and pitfalls of population-wide studies implementing faecal sampling for gut microbiome research.
Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife ...conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment.
Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.
Mass spectrometry imaging (MSI) has become a widespread analytical technique to perform nonlabeled spatial molecular identification. The Achilles' heel of MSI is the annotation and identification of ...molecular species due to intrinsic limitations of the technique (lack of chromatographic separation and the difficulty to apply tandem MS). Successful strategies to perform annotation and identification combine extra analytical steps, like using orthogonal analytical techniques to identify compounds; with algorithms that integrate the spectral and spatial information. In this review, we discuss different experimental strategies and bioinformatics tools to annotate and identify compounds in MSI experiments. We target strategies and tools for small molecule applications, such as lipidomics and metabolomics. First, we explain how sample preparation and the acquisition process influences annotation and identification, from sample preservation to the use of orthogonal techniques. Then, we review twelve software tools for annotation and identification in MSI. Finally, we offer perspectives on two current needs of the MSI community: the adaptation of guidelines for communicating confidence levels in identifications; and the creation of a standard format to store and exchange annotations and identifications in MSI.
With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, ...detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.
The state-of-the-art of sampling procedures is specified in appropriate guidelines, but there is a permanent need to minimise the risk to the water sample quality, in particular for waters with ...specific characteristics such as geothermal waters. Improper sampling leads to erroneous results and, consequently, e.g. increased uncertainty in models of geochemical processes (scaling, corrosion) in geothermal installations, aquifer geothermometry, or environmental assessments. The impact of the applied filters (nylon, PVDF, MCE), containers (HDPE, borosilicate glass) and preservation on concentration of 34 major, minor and trace elements was studied for the samples of two geothermal waters used in practice. The results of replicated chemical analyses in duplicated samples of geothermal waters and deionized water blanks were subjected to statistical analyses, including nonparametric tests (Kruskal-Wallis, Dunn's, Kolmogorov-Smirnov, Mann-Whitney U, Wald-Wolfowitz). The PVC housing of the used MCE syringe filters contaminates water samples with Sb. Borosilicate glass increases Al, Cu, Pb and Se concentrations in samples. Acidification protects against changes in concentrations of numerous elements (i.a. Al, B, Ba, Cu, Fe, Ni, Pb) in the solution. Results of analysis performed indicated that using nylon membrane filter and HDPE (properly precleaned to wash out extractables) containers for thermal water sampling is recommended.
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•New broad study on the effects of sampling on the geothermal water samples quality.•High-quality borosilicate glass might affect numerous elements in water sample.•Housing material of the MCE syringe filters contaminates geothermal water samples.•Nylon filters and HDPE containers recommended for thermal water sampling.•Water sampling protocols require thorough and regular verification by analytical tests.
Purpose
According to standard procedure recommended by the Water Framework Directive (WFD), dissolved concentrations of potentially toxic elements (PTEs) in river water are determined by inductively ...coupled plasma mass spectrometry (ICP-MS) in filtered (0.45 µm) and acidified (pH 2) samples. Properly prepared and stored composite samples can enhance the temporal representativity of monitoring without increasing analytical costs. For this purpose, the WFD recommends freezing, which can preserve the species integrity and prevent adsorption processes of PTEs.
Methods
Low storage temperature in hard water samples can trigger precipitation of calcium carbonate (CaCO
3
) and subsequent co-precipitation of PTEs. To test and determine to what extent co-precipitation with CaCO
3
can influence the determination of PTE concentrations, composite river water samples from two case study catchments in Hungary (Zagyva and Koppány) were prepared following two different sample preservation procedures. To study the behavior of PTEs in river water during storage, in the first procedure, samples were frozen, and they were thawed, filtered, and acidified directly prior to the analysis. In the second procedure, samples were filtered on-site and acidified prior to freezing to prevent precipitation of CaCO
3
and then only thawed to carry out the chemical analyses. Concentrations of PTEs were determined by ICP-MS.
Results
A statistical evaluation of the results using Student’s
t
-test revealed significant differences between the two sample preservation procedures, suggesting that PTEs were largely co-precipitated with CaCO
3
if the samples were not acidified prior to freezing.
Conclusion
When establishing protocols for sample preservation procedures, the phenomenon of co-precipitation of PTEs with CaCO
3
should be considered if the samples were not acidified before freezing. Therefore, to prevent co-precipitation of PTEs with CaCO
3
, samples should be filtered and acidified before freezing.
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