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.
OBJECTIVES: Stability of concentrations of urinary stone-related metabolites was analyzed from samples of recurrent urinary stone formers to assess necessity and effectiveness of urine acidification during collection and storage. METHODS: First-morning urine was collected from 20 adult calcium-stone forming patients at Tomas Bata Hospital in the Czech Republic. Urine samples were analyzed for calcium, magnesium, inorganic phosphate, uric acid, sodium, potassium, chloride, citrate, oxalate, and urine particles. The single-voided specimens were collected without acidification, after which they were divided into three groups for storage: samples without acidification ("NON"), acidification before storage ("PRE"), or acidification after storage ("POST"). The analyses were conducted on the day of arrival (day 0, "baseline"), or after storage for 2 or 7 days at room temperature. The maximum permissible difference (MPD) was defined as ±20 % from the baseline. RESULTS: The urine concentrations of all stone-related metabolites remained within the 20 % MPD limits in NON and POST samples after 2 days, except for calcium in NON sample of one patient, and oxalate of three patients and citrate of one patient in POST samples. In PRE samples, stability failed in urine samples for oxalate of three patients, and for uric acid of four patients after 2 days. Failures in stability often correlated with high baseline concentrations of those metabolites in urine. CONCLUSIONS: Detailed procedures are needed to collect urine specimens for analysis of urinary stone-related metabolites, considering both patient safety and stability of those metabolites. We recommend specific preservation steps.
- MeSH
- Urinalysis methods MeSH
- Adult MeSH
- Hydrogen-Ion Concentration MeSH
- Uric Acid urine MeSH
- Middle Aged MeSH
- Humans MeSH
- Urinary Calculi * urine MeSH
- Specimen Handling methods MeSH
- Recurrence * MeSH
- Urine Specimen Collection methods MeSH
- Aged MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Drug Resistance, Microbial MeSH
- Antigens analysis MeSH
- Diagnostic Techniques, Respiratory System classification MeSH
- Molecular Diagnostic Techniques MeSH
- Respiratory Tract Infections * diagnosis etiology microbiology MeSH
- Culture Techniques methods MeSH
- Humans MeSH
- Microbiological Techniques * classification methods MeSH
- Microscopy methods MeSH
- Specimen Handling methods MeSH
- Blood Specimen Collection MeSH
- Antibodies analysis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
The MBT Pathfinder is an automated colony-picking robot designed for efficient sample preparation in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This article presents results from three key experiments evaluating the instrument's performance in conjunction with MALDI Biotyper instrument. The method comparison experiment assessed its clinical performance, demonstrating comparable results with gram-positive, gram-negative, and anaerobic bacteria (scores larger than 2.00) and superior performance over simple direct yeast transfer (score: 1.80) when compared to samples prepared manually. The repeatability experiment confirmed consistent performance over multiple days and labs (average log score: 2.12, std. deviation: 0.59). The challenge panel experiment showcased its consistent and accurate performance across various samples and settings, yielding average scores between 1.76 and 2.19. These findings underline the MBT Pathfinder as a reliable and efficient tool for MALDI-TOF mass spectrometry sample preparation in clinical and research applications.
- MeSH
- Bacteria * classification isolation & purification MeSH
- Automation, Laboratory * methods MeSH
- Humans MeSH
- Specimen Handling methods MeSH
- Reproducibility of Results MeSH
- Robotics * MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
Osteosarcoma and Ewing sarcoma are bone tumors mostly diagnosed in children, adolescents, and young adults. Despite multimodal therapy, morbidity is high and survival rates remain low, especially in the metastatic disease setting. Trials investigating targeted therapies and immunotherapies have not been groundbreaking. Better understanding of biological subgroups, the role of the tumor immune microenvironment, factors that promote metastasis, and clinical biomarkers of prognosis and drug response are required to make progress. A prerequisite to achieve desired success is a thorough, systematic, and clinically linked biological analysis of patient samples, but disease rarity and tissue processing challenges such as logistics and infrastructure have contributed to a lack of relevant samples for clinical care and research. There is a need for a Europe-wide framework to be implemented for the adequate and minimal sampling, processing, storage, and analysis of patient samples. Two international panels of scientists, clinicians, and patient and parent advocates have formed the Fight Osteosarcoma Through European Research consortium and the Euro Ewing Consortium. The consortia shared their expertise and institutional practices to formulate new guidelines. We report new reference standards for adequate and minimally required sampling (time points, diagnostic samples, and liquid biopsy tubes), handling, and biobanking to enable advanced biological studies in bone sarcoma. We describe standards for analysis and annotation to drive collaboration and data harmonization with practical, legal, and ethical considerations. This position paper provides comprehensive guidelines that should become the new standards of care that will accelerate scientific progress, promote collaboration, and improve outcomes.
- MeSH
- Biological Specimen Banks MeSH
- Sarcoma, Ewing * therapy pathology diagnosis MeSH
- Humans MeSH
- Biomarkers, Tumor MeSH
- Bone Neoplasms * therapy pathology MeSH
- Specimen Handling * methods standards MeSH
- Osteosarcoma * therapy pathology diagnosis MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Geographicals
- Europe MeSH
- MeSH
- Antifungal Agents administration & dosage pharmacology therapeutic use MeSH
- Dermatomycoses diagnosis etiology drug therapy MeSH
- Candidiasis diagnosis etiology drug therapy MeSH
- Humans MeSH
- Mycoses * diagnosis etiology drug therapy MeSH
- Eye Infections, Fungal diagnosis etiology drug therapy MeSH
- Specimen Handling methods MeSH
- Otomycosis diagnosis etiology drug therapy MeSH
- Lung Diseases, Fungal diagnosis etiology drug therapy MeSH
- Risk Factors MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
Proper fixing and long-term preservation of entomological evidence are essential in collections and research and crucial in applied fields such as forensic entomology. Incorrectly stored samples may lose important morphological features over time, rendering molecular analyses exceedingly difficult. The most effective method for preserving soft samples such as larvae is fluid preservation. It uses a combination of a wide range of fixatives and storage fluids. However, very little comparative work has been done to determine the effects of long-term storage on sample quality in terms of color, shape, and DNA stability. Moreover, the current golden standard in forensic entomology has been tailored for age estimation of larvae of Diptera, which differ from larvae of Coleoptera in morphology and subsequently in applied methods. We compared the effects of combinations of 6 commonly used fixatives and 6 commonly used storage fluids on midsized larvae of the forensically important beetle, Necrodes littoralis (Linnaeus, 1758), in terms of color, shape, and suitability for DNA analyses over a 2-yr period. We were looking for combinations that can preserve specimens in a satisfactory state, can be used on a regular basis, do not require advanced protection or skills of the personnel, and are not toxic or too harmful to the environment. We found not only several methods that scored significantly better in the tested parameters compared with the golden standard but also several common methods that should be avoided. The effects of agents on each tested category are discussed in detail.
- MeSH
- Color MeSH
- Coleoptera * MeSH
- Time Factors MeSH
- DNA * analysis MeSH
- Forensic Entomology methods MeSH
- Larva * growth & development MeSH
- Preservation, Biological methods MeSH
- Specimen Handling methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
BACKGROUND: Testing of pooled samples is an effective strategy for increasing testing capacity while saving resources and time. This study aimed to validate pooled testing and gather real-life data on its use for Covid-19 surveillance with a gargle lavage (GL) self-sampling strategy. METHODS: Two-stage pooled testing with pools of 6 and 12 samples was used for preventive testing of an asymptomatic population and Covid-19 surveillance in Czech schools. Both GL and nasopharyngeal swabs were used for sampling. RESULTS: In total, 61,111 samples were tested. The use of pooled testing for large-scale Covid-19 surveillance reduced consumable costs by almost 75% and increased testing capacity up to 3.8-fold compared to standard methods. RT-PCR experiments revealed a minimal loss of sensitivity (0-2.2%) when using pooled samples, enabling the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes with Ct values >35. The minor loss of sensitivity was counterbalanced by a significantly increased throughput and the ability to substantially increase testing frequencies. CONCLUSIONS: Pooled testing is considerably more cost-effective and less time-consuming than standard testing for large-scale Covid-19 surveillance even when the prevalence of SARS-CoV-2 is fluctuating. Gargle lavage self-sampling is a non-invasive technique suitable for sample collection without a healthcare worker's assistance.
- MeSH
- COVID-19 * diagnosis epidemiology MeSH
- Humans MeSH
- Nasopharynx * virology MeSH
- Specimen Handling * methods MeSH
- SARS-CoV-2 * genetics isolation & purification MeSH
- Sensitivity and Specificity MeSH
- COVID-19 Nucleic Acid Testing methods MeSH
- COVID-19 Testing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
One of the least invasive sampling methods suitable for self-sampling is saliva spitting. The aim of this study is to evaluate the suitability of saliva self-sampling for unsupervised testing. Two self-sampling strategies were compared on the basis of visual evaluation of samples, measurement of cortisol levels in samples and questionnaire survey. The saliva samples obtained by supervised self-sampling were found to be fully suitable for further analysis. In contrast, not all saliva samples obtained from unsupervised self-collection can be used: 13% non-compliance with the minimum required sample volume, 8% with some food/drink residues and 26% taken at the wrong day time. About 42% of the unsupervised probands made at least one significant error in the saliva self-collection procedure. These results indicate that the accuracy of the results based on the analysis of samples received from saliva self-sampling is limited. For clinical investigation, the presence of an inner standard (referring to the reliability of the sampling procedure) is required.
One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.
- MeSH
- Cell Differentiation physiology MeSH
- Adult MeSH
- Genetic Vectors genetics MeSH
- Induced Pluripotent Stem Cells * physiology transplantation MeSH
- Rats MeSH
- Humans MeSH
- Spinal Cord MeSH
- Brain MeSH
- Neural Stem Cells * physiology transplantation MeSH
- Specimen Handling methods MeSH
- Tissue and Organ Harvesting methods MeSH
- Swine MeSH
- Cellular Reprogramming * genetics physiology MeSH
- Graft Survival physiology MeSH
- Injections, Spinal * adverse effects instrumentation methods MeSH
- Stem Cell Transplantation * adverse effects instrumentation methods MeSH
- Sendai virus MeSH
- Treatment Outcome MeSH
- Animals MeSH
- Check Tag
- Adult MeSH
- Rats MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
