Sample Preparation Time Research Articles

Endocannabinoid and steroid analysis in infant and adult nails by LC–MS/MS

A common method to quantify chronic stress is the analysis of stress markers in keratinized matrices such as hair or nail. In this study, we aimed to validate a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the combined quantification of steroid hormones and endocannabinoids (eCBs) in the keratinized matrix nail. Furthermore, we aimed to investigate the suitability of the nail matrix for the detection of these stress markers in a pilot study. An LC–MS/MS method was used for the simultaneous identification and quantification of four eCBs (2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA), palmitoylethanolamide (PEA)) and five steroid hormones (cortisol, cortisone, androstenedione, progesterone, testosterone) in human nails using a surrogate analyte method for each analyte. The method was validated in terms of selectivity, response factor, linearity, limit of quantification (LOQ), precision, accuracy, matrix effect, recovery, robustness, and autosampler stability. Nail samples were extracted for 1 h with methanol following a clean-up with a fully automated supported liquid extraction (SLE). The influence of nail weight on the quantification was investigated by using 0.5–20 mg of nail sample. As a proof of concept, nail samples (N = 57) were analyzed from a cohort representing newborns (1 month old), children (between 1 and 10 years), and adults (up to 43 years). It could be shown that the established workflow using a 1 hour extraction and clean-up by SLE was very robust and resulted in a short sample preparation time. The LC–MS/MS method was successfully validated. Matrix effects with ion enhancement occurred mainly for 2-AG. Sample weights below 5 mg showed variations in quantification for some analytes. Certain analytes such as PEA and progesterone could be accurately quantified at a sample weight lower than 5 mg. This is the first study where steroids and eCBs could be simultaneously detected and quantified in infant and adult nails. These results show that nails may serve as an alternative keratinized matrix (compared to hair) for the retrospective monitoring of cumulative eCB and steroid hormone levels. The combined assessment of eCBs and steroids from nails could provide a new approach to gain new insights into stress exposure in newborns and adults.Graphical abstract

High-throughput analysis for the simultaneous quantification of nine beta-lactam antibiotics in human plasma by UPC2-MS/MS: Method development, validation, and clinical application

Quantification of beta-lactam antibiotics can be performed by using liquid chromatography in combination with tandem mass spectrometry (MS/MS) or ultraviolet (UV) detection. Since beta-lactam antibiotics are known as highly polar analytes, using standard reversed phase chromatography will result in very early elution, which is often not desirable. Some retention is preferred to reduce matrix effects, because a high amount of non-retained molecular matrix species elute early from the column. For highly polar analytes, ultra-performance convergence chromatography (UPC2) may be a suitable alternative. This method is based on supercritical fluid chromatography. To our knowledge, we developed the first UPC2-MS/MS method for the determination of amoxicillin, benzylpenicillin, flucloxacillin, piperacillin, cefotaxime, cefuroxime, ceftazidime, imipenem, meropenem, and the free fraction of cefuroxime and flucloxacillin in human plasma. The method was validated according to the Food and Drug Administration guidelines. The method was found linear (r2 >0.990) for all analytes. The inaccuracies and imprecisions were < 15% for all analytes. The matrix effect and recovery were nearly all consistent with coefficient of variation of less than 15% and no significant carryover effect was observed. Furthermore, this method was found to be suitable for daily routine analysis in hospital settings, requiring only 50 µL of plasma. This novel, sensitive, and specific UPC2-MS/MS method demonstrated its value in the analysis of a more than 800 human plasma samples in a clinical trial using simple and fast sample preparation and short analysis run time of only 5 min

Assessment of the Effect of Soil Sample Preparation, Water Content and Excitation Time on Proximal X-ray Fluorescence Sensing.

X-ray fluorescence (XRF) spectroscopy offers a fast and efficient method for analysing soil elemental composition, both in the laboratory and the field. However, the technique is sensitive to spectral interference as well as physical and chemical matrix effects, which can reduce the precision of the measurements. We systematically assessed the XRF technique under different sample preparations, water contents, and excitation times. Four different soil samples were used as blocks in a three-way factorial experiment, with three sample preparations (natural aggregates, ground to ≤2 mm and ≤1 mm), three gravimetric water contents (air-dry, 10% and 20%), and three excitation times (15, 30 and 60 s). The XRF spectra were recorded and gave 540 spectra in all. Elemental peaks for Si, K, Ca, Ti, Fe and Cu were identified for analysis. We used analysis of variance (anova) with post hoc tests to identify significant differences between our factors and used the intensity and area of the elemental peaks as the response. Our results indicate that all of these factors significantly affect the XRF spectrum, but longer excitation times appear to be more defined. In most cases, no significant difference was found between air-dry and 10% water content. Moisture has no apparent effect on coarse samples unless ground to 1 mm. We suggested that the XRF measurements that take 60 s from dry samples or only slightly moist ones might be an optimum option under field conditions.

Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays

Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5′-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.

A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing.

A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army’s Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology’s Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

Rapid Antibiotic Susceptibility Testing by Deuterium Labeling of Bacterial Lipids in On-Target Microdroplet Cultures.

Antimicrobial resistance is a serious challenge facing human and veterinary health. Current methods of detecting resistance are limited in turn-around time or universal detection. In this work, a new antimicrobial susceptibility test is developed and validated, which utilizes deuterium labeling of membrane lipids to track the growth of bacterial cells. We hypothesize that deuterium uptake and subsequent labeling of lipids can be detected using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Additionally, bacteria growth is performed on the MALDI target, minimizing sample preparation materials and time. When two Escherichia coli strains are grown in the presence of deuterium oxide, labeling can be detected in as little as 30 min to 2 h. The labeling efficiency, or the ratio of labeled to unlabeled lipid peaks, provides information about the growth rate of bacteria. This growth ratio can differentiate between resistant and susceptible strains of bacteria as a resistant strain will maintain ∼50% labeling efficiency between untreated and treated cultures. In comparison, a susceptible strain will see a decrease in fractional abundance of deuterium from ∼50% in the untreated to ∼10% in the treated. This approach is applied to measure the minimum inhibitory concentration (MIC) of the resistant and susceptible strains from on-target microdroplet culture in a range of antibiotic concentrations. The first antibiotic concentration with a significant decrease in fractional abundance of deuterium correlates well with a traditionally obtained MIC using broth dilution, indicating the clinical relevance of the results.

Determination of in situ hydrocarbon contents in shale oil plays. Part 1: Is routine Rock–Eval analysis reliable for quantifying the hydrocarbon contents of preserved shale cores?

An accurate determination of in situ hydrocarbon (oil) content is critical for the assessment of shale oil plays in terms of their total resources in place and recovery potential. Although correction of light hydrocarbon loss to the Rock–Eval S1 parameters on conventional core and cuttings samples has been recently attempted using preserved (e.g., sealed or pressure–preserved) cores, hydrocarbon evaporation loss during sample preparation and delay time before the actual commencement of Rock–Eval analysis has not received sufficient attention. In this study, a combination of petrophysical and geochemical techniques including Dean–Stark extraction, hydrogen nuclear magnetic resonance (1H-NMR) T1-T2 mapping, thermal desorption–gas chromatography (TD–GC) analysis, and Rock–Eval programmed pyrolysis were used in parallel on a set of 30 preserved shale cores to determine their total hydrocarbon contents. The preserved shale cores were analyzed in large pieces (e.g., 3–5 cm in size) by the former two petrophysical methods while using fine powder (e.g., <60 mesh) by the latter two thermal analytical methods. The total free hydrocarbon contents (equivalent of Rock–Eval S1) determined on the studied lacustrine shale samples with a Ro ranging from 1.00% to 1.51% followed the order of Dean–Stark (average 10.27 mg/g) ≈ NMR (10.34) ＞ TD–GC (6.65) ＞ Rock–Eval (3.93). The core crushing and grinding process required for both TD-GC and Rock–Eval pyrolysis analyses was found to cause a loss of approximately 35% to the in situ hydrocarbons. In addition, Rock–Eval analysis delay time (of 5 min) could lead to another 25% reduction to the total free hydrocarbons. In combination, sample preparation and analysis delay resulted in up to 60% reduction to the Rock–Eval S1 peaks for the preserved shale cores, and a 30% loss to the liquid (C6+) hydrocarbons in particular. To the best of our knowledge, this is the first reported observation that in situ light hydrocarbon contents can be severely underestimated by Rock–Eval and TD–GC analyses using powdered forms of preserved core samples. The extents of light hydrocarbon loss in conventional cores have been underestimated using the Rock–Eval S1 difference between preserved shale and its long–duration exposed replicates. Hence, when quantifying the hydrocarbon contents of the preserved shale samples, techniques utilizing larger rock pieces are highly recommended.

Determination of Picogram-per-Gram Concentrations of 231Pa and 230Th in Sediments by Melt Quenching and Laser Ablation Mass Spectrometry.

Uranium, thorium, and protactinium radionuclides in marine sediments are important proxies for understanding the earth's environmental evolution. Conventional solution-based methods, which typically involve isotope spike preparation, concentrated acid sample digestion, column chemistry, and mass spectrometry, allow precise but time-consuming and costly measurements of these nuclide concentrations (i.e., 230Th and 231Pa). In this work, we have established an efficient method for 230Th and 231Pa measurement of marine sediments down to the picogram-per-gram level without purification and enrichment. Our method first transforms a small amount of thermally decomposed sediments (∼0.1-0.2 g) to homogeneous silicate glass using a melt quenching technique and then analyzes the glass with laser ablation multicollector inductively coupled plasma-mass spectrometry. Standard sample bracketing with isotope-spike-calibrated glass standards prepared in this study was used to correct for instrumental fractionation during measurement. It is demonstrated that our method can accurately determine the U-Th-Pa concentrations of typical marine sediments in the late Pleistocene with precision of a few percent. Compared with the conventional solution-based methods, the turnover time of sample preparation and measurement with our established protocol is greatly reduced, facilitating future application of U-series radionuclides in reconstructing oceanic processes at high temporal and spatial resolution.

Investigating the potential of rock surface burial dating using IRPL and IRSL imaging

Techniques for spatially resolved measurements of infrared-stimulated luminescence (IRSL) and novel Infrared-photoluminescence (IRPL) emissions have recently been developed for applications of rock surface dating. Such spatially resolved measurements overcome the need for separating out mineral fractions, speed-up sample preparation and measurement times, and data can be quickly processed provide high-resolution luminescence-depth profiles. Here, we investigate the potential of using spatially resolved IRPL and IRSL measurements for rock surface burial dating using two large (cm-scale) rock samples with controlled exposure and surface dose histories. We use a SAR style measurement protocol, with a test-dose normalisation step to monitor sensitivity changes, a preheat to remove unstable charges and a bleaching step to reset the IRPL signal. Through establishing the response of IRPL and IRSL to dose, we are able to construct 2D maps of equivalent doses (Des) for each sample. The results here indicate that spatially resolving IRSL and IRPL from large rock samples has the potential to be used for rock surface burial dating and offers a means to investigate the spatial distribution of dose and mineral-dependent sensitivity changes through cm-scale rock samples.

A streamlined method for the fast and cost-effective detection of bacterial pathogens from positive blood cultures for the BacT/ALERT blood culture system using the Vitek MS mass spectrometer

Background and objectivePrompt pathogen identification of blood stream infections is essential to provide appropriate antibiotic treatment. Therefore, the objective of this prospective single centre study was to establish an inexpensive, fast and accurate protocol for bacterial species identification with SDS protein-extraction directly from BacT/Alert® blood culture (BC) bottles by VitekMS®.ResultsCorrect species identification was obtained for 198/266 (74.4%, 95%-CI = [68.8%, 79.6%]) of pathogens. The protocol was more successful in identifying 87/96 (91.4%, 95%-CI = [83.8%, 93.2%]) gram-negative bacteria than 110/167 (65.9%, 95%-CI = [58.1%, 73.0%]) gram-positive bacteria. The hands-on time for sample preparation and measurement was about 15 min for up to five samples. This is shorter than for most other protocols using a similar lysis-centrifugation approach for the combination of BacT/Alert® BC bottles and the Vitek® MS mass spectrometer. The estimated costs per sample were approx. 1.80€ which is much cheaper than for commercial kits.ConclusionThis optimized protocol allows for accurate identification of bacteria directly from blood culture bottles for laboratories equipped with BacT/Alert® blood culture bottles and VitekMS® mass spectrometer.

Development of a robust 30-minute reverse-phase high pressure liquid chromatography method to measure amino acids using widely available equipment and its comparison to current clinical ion-exchange chromatography measurement

We have developed a fast and accurate method that uses a small volume of sample to determine over 25 of the typically reported amino acids in human plasma. Samples were prepped with a single step using a spin filter to remove proteins, avoiding the decreased sensitivity from dilution in acid precipitation. Using a reverse phase (RP) High Performance Liquid Chromatography (HPLC) system with O-phthaldehyde (OPA) as the pre-column derivatization reagent, and UV detection at 338 nm, we did a direct comparison with the most common ion exchange/ninhydrin method used in clinical labs on the same plasma samples with 95% concurrence, analysis of amino acid standard solutions returned 99% concurrence. With a sample preparation time of 30 min, utilizing less than 25 μl of sample and with a chromatography run of 30 min, this method can substantially increase access to analysis in both clinical and research laboratories using instruments that are more widely available. SynopsisWe describe a rapid and easily deployed method for sensitive amino measurement in biological samples.

Sample preparation and quantification of polar drug, allopurinol, in human plasma using LCMSMS.

A fast, selective and reproducible LC-MS/MS method with simple sample preparation was developed and validated for a polar compound, allopurinol in human plasma, using acyclovir as internal standard (IS). Chromatographic separation was achieved using Agilent Poroshell 120 EC-C18 (100 × 2.1 mmID, 2.7 µm) analytical column. The mobile phase was comprised of 0.1%v/v formic acid-methanol (95:05; v/v), at a flow rate of 0.45 mL/min. The effect of different protein precipitation agents used in sample preparation such as methanol, acetonitrile, a mixture of acetonitrile-methanol and a mixture of acetonitrile-acetone were evaluated to optimize the extraction efficiency of allopurinol and IS. The use of acetone-acetonitrile (50:50, v/v) as protein precipitating agent shortened the sample preparation time and improved the recovery of allopurinol to above 93%. The IS-normalised matrix factors at two concentration levels were 1.0, with CV of 5.1% and 4.2%. Allopurinol in plasma was stable at benchtop for 24 h, in autosampler tray for 48 h, in instrumentation room for 48 h, in freezer after 7 freeze-thaw cycles and in freezer for 140 days. Allopurinol stock standard solutions were stable for 140 days at room temperature and in the chiller. The short sample run time of the validated bioanalytical method allowed high throughput analysis of plasma samples in pharmacokinetic study of an allopurinol formulation. The robustness and reproducibility of the bioanalytical method was reaffirmed through incurred sample reanalysis (ISR).

Near-infrared techniques for fraud detection in dairy products: A review.

The dairy products sector is an important part of the food industry, and their consumption is expected to grow in the next 10 years. Therefore, the authentication of these products in a faster and precise way is required for the sake of public health. This review proposes the use of near-infrared techniques for the detection of food fraud in dairy products as they are faster, nondestructive, environmentally friendly, do not require sample preparation, and allow multiconstituent analysis. First, we have described frequent forms of food fraud in dairy products and the application of traditional techniques for their detection, highlighting gaps and counterproductive characteristics for the actual global food chain, as longer sample preparation time and use of reagents. Then, the application of near-infrared spectroscopy and hyperspectral imaging for the detection of food fraud mainly in cheese, butter, and yogurt are described. As these techniques depend on model development, the coverage of different dairy products by the literature will promote the identification of food fraud in a faster and reliable way.

Simultaneous Determination of Multi-Class Pesticide Residues and PAHs in Plant Material and Soil Samples Using the Optimized QuEChERS Method and Tandem Mass Spectrometry Analysis.

New analytical approaches to the simultaneous identification and quantification of 94 pesticides and 13 polycyclic aromatic hydrocarbons (PAHs) in five representative matrices (pepper, apple, lettuce, wheat, and soil) were developed. The analyses were based on gas chromatography coupled with triple quadrupole tandem mass spectrometry (GC-MS/MS). The procedure was optimized by changing the solvent used during the extraction, from acetonitrile to the acetone: n-hexane mixture at a volume ratio of 1:4 (v/v), as well as the use of a reduced amount of water during the extraction of compounds from cereals. An additional modification was the use of florisil instead of GCB in the sample cleanup step. A full method validation study was performed, at two concentration levels (LOQ and 1000 × LOQ), which showed satisfactory results for all analytes from the PAHs group, with recoveries ranging from 70.7–115.1%, and an average RSD of 3.9%. Linearity was tested in the range of 0.001–1.000 mg/kg and showed coefficients of determination (R2) ≥ 0.99 for all PAHs. Satisfactory recovery and precision parameters (LOQ and 100 × LOQ) were achieved for almost all analytes from the pesticide group in the range of 70.1–119.3% with the mean RSD equal to 5.9%. The observed linearity for all analytes in the concentration range of 0.005–1.44 mg/kg was R2 ≥ 0.99, with the exception of famoxadone, chizalofop-p-ethyl, prothioconazole, spirodiclofen, tefluthrin, and zoxamid. The extended uncertainties were estimated, using a top-down approach of 9.9% (average) and 15.3% (average) for PAHs and pesticides samples, respectively (the coverage factor k = 2, the 95% confidence level). Ultimately, the method was successfully applied to determine pesticide residues in commercial samples of fruit, vegetables and grain, and soil samples for PAHs, which were collected from selected places in the Podkarpacie region. A total of 38 real samples were tested, in which 10 pesticides and 13 PAHs were determined. Proposed changes allow us to shorten the sample preparation time (by 20%) and to reduce the consumption of organic solvents (by 17%). The use of florisil for sample cleanup, instead of GCB, improves the recovery of compounds with flat particles.

Development of a rapid and sensitive LC-MS/MS assay for the quantification of commonly abused drugs in Asia in a micro-segment of a single hair using microwave-assisted extraction and dansyl chloride derivatization

A high-throughput method using microwave-assisted extraction, chemical derivatization and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of illicit drugs and their metabolites, including amphetamine (AMP), methamphetamine (MA), methylenedioxy- amphetamine (MDA), methylenedioxy-methamphetamine (MDMA), morphine (MOR), 6-acetylmorphine (6-AM), ketamine (K), and norketamine (NK) in a micro-segment of a single hair (0.4 mm). In order to elevate the throughput and sensitivities of selected compounds, 3 min microwave-assisted extraction and 10 min derivatization with dansyl chloride (DC) were employed. After derivatization, all compounds except ketamine and norketamine were derivatized and enhanced the sensitivities significantly. Derivatives showed intense fragment ions and low background noise on DC-MOR, DC-6-AM, and four DC-amphetamine-type stimulants. The total sample preparation and analysis time was 50 min. The calibration range was from LOQ to 5000 pg/mg, the coefficient of determination was better than 0.997. Intra-assay precision and accuracy were generally less than 15%. Limits of detection ranged from 15 to 50 pg/mg, limits of quantitation ranged between 45 and 125 pg/mg. The matrix effect was better than 90%. The method was successfully applied to the analysis of actual hair samples collected from multi-drug abusers. This advanced method showed practicality in hair analysis and was suitable for the extremely insufficient sample.

DirectDetect SARS-CoV-2 Direct Real-Time RT-PCR Study Using Patient Samples.

COVID-19 is an infectious disease that caused a global pandemic affecting people worldwide. As disease detection and vaccine rollout continue to progress, there is still a need for efficient diagnostic tools to satisfy continued testing needs. This preliminary study evaluated a novel SARS-CoV-2 diagnostic test called DirectDetect SARS-CoV-2 Direct Real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on a limited sample size of 24 respiratory samples from 14 SARS-CoV-2-positive patients. The test is advantageous compared to others on the market since it does not require viral transport medium or viral RNA extraction prior to nucleic acid amplification and detection. This capability transforms the hours-long sample preparation time into a minutes-long procedure while also eliminating the need for many costly reagents which may be difficult to obtain during the surge in nucleic acid-based testing during the pandemic. The results show a positive agreement of 94.7, 100, and 94.7% between dry sample swabs, treated samples, and untreated samples tested using the DirectDetect SARS-CoV-2 Direct Real-time RT-PCR compared to tests used in a clinical laboratory, respectively. The findings indicate that DirectDetect can be used for multiple different sample types while reducing the number of reagents and time needed for diagnosis. Although this study shows promising results using the DirectDetect results, further validation of this test using a larger sample set is required to assess the true performance of this test.

Rapid in situ assessment of luminescence-bleaching depths for deriving burial and exposure chronologies of rock surfaces

Recent developments in luminescence dating offer new ways to date exposure and burial durations of rocks. The new rock surface dating methods ideally require high-resolution data, faster sample preparation and measurement times, and field screening methods to select samples with appropriate luminescence characteristics and bleaching histories. Presented here is a demonstration of an EMCCD (electron multiplying charge coupled device) based system capable of imaging high-resolution infrared stimulated luminescence (IRSL) and infrared-photoluminescence (IRPL) from rock samples. The IRPL can be detected at both 880 nm and 955 nm. Using this instrument, the entire luminescence-depth profile can be reconstructed by imaging a single surface cut perpendicular to the exposed rock face. We demonstrate the possibility of reconstructing luminescence-depth profiles suitable for rock surface dating from large (cm-scale) rock samples, without using a regeneration dose for normalisation of the natural luminescence signals. Based on the different bleaching characteristics of the IRSL and IRPL emissions at 880 nm and 955 nm, we show that it is possible to gain reliable estimates of bleaching depths from measurement of as few as two images of the IRPL signal (one for each emission), or from measurement of the IRSL decay curve. We thus by-pass laborious sample preparations and the need for a gamma source to estimate the bleaching depth, thereby extending the 2D luminescence-depth profile imaging technique to other laboratories that lack access to ionising radiation facilities. This study also makes a significant progression towards development of a field instrument for in situ relative exposure dating, and sample screening for rock surface burial dating. • Direct imaging of luminescence-depth profiles in rocks using IRPL or IRSL. • New methods for normalising spatial variations in luminescence without irradiation. • Characterising bleachability of different OSL emissions from the principal trap. • Rock surface dating for rapid field analysis or laboratories lacking irradiation facilities.

Development and evaluation of handy jute fibre bundle strength tester

Jute is traditionally tested for bundle strength by breaking the fibers in the hand and using a manual bundle strength tester. Hand method is subjective and grade may vary from grader to grader. The manual bundle strength tester requires a lot of time for sample preparation and manual calculation of the strength of the fibre. In the present study, the goal was to develop an accurate and quick tester for measuring the strength of jute fibre bundles. The unit consists of a handle, fibre holder with clamp, pointer with an analog indicator, body, and a chain. The analog indicator has a colour code to indicate the strength of the fibre. This instrument has an overall dimension of 350 × 185 × 90 mm. Randomly twenty samples were selected from the bale sample to perform the test. The results of the developed instrument were compared with the results of a manual bundle strength tester. The T-test result showed that there was no significant (p &gt; 0.05) difference between results from the handy type bundle strength tester and mechanical instrument. Strength of the jute measured from a handy jute fibre tester was almost on par with manual type instrument. The repeatability of the handy bundle strength tester at 95% confidence level is 3.02 and 2.90 g/tex, respectively for Corchorus olitorius and Corchorus capsularis . The instrument readings are repeatable since the difference between two readings is less than the r p (repeatability) value at 95% confidence level. The study concluded that the time taken to measure bundle strength of jute was shorter than with a manual tool. In contrast to manual instruments, this instrument is easy to operate and can provides reliable results.

Imaging the Kidney with an Unconventional Scanning Electron Microscopy Technique: Analysis of the Subpodocyte Space in Diabetic Mice.

Transmission electron microscopy (TEM) remains the gold standard for renal histopathological diagnoses, given its higher resolving power, compared with light microscopy. However, it imposes several limitations on pathologists, including longer sample preparation time and a small observation area. To overcome these, we introduced a scanning electron microscopy (SEM) technique for imaging resin-embedded semi-thin sections of renal tissue. We developed a rapid tissue preparation protocol for experimental models and human biopsies which, alongside SEM digital imaging acquisition of secondary electrons (SE–SEM), enables fast electron microscopy examination, with a resolution similar to that achieved by TEM. We used this unconventional SEM imaging approach to investigate the subpodocyte space (SPS) in BTBR ob/ob mice with type 2 diabetes. Analysis of semi-thin sections with secondary electrons revealed that the SPS had expanded in volume and covered large areas of the glomerular basement membrane, forming wide spaces between the podocyte body and the underlying filtering membrane. Our results show that SE–SEM is a valuable tool for imaging the kidney at the ultrastructural level, filling the magnification gap between light microscopy and TEM, and reveal that in diabetic mice, the SPS is larger than in normal controls, which is associated with podocyte damage and impaired kidney function.

Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device.

It is well known that hydrogen peroxide (H2O2) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H2O2, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H2O2. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H2O2-sensing device has allowed the detection of H2O2 in the range of 0.5-60 μM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H2O2 release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 μL), and not the cells, is required.