Standard Microscopy Research Articles

Glutaraldehyde-enhanced autofluorescence as a general tool for 3D morphological imaging.

Routine histochemical techniques are capable of producing vast amount of information from diverse samples types, but these techniques are limited in their ability to generate 3D information. Autofluorescence imaging can be used to analyse samples in 3D but it suffers from weak/low signal intensities. Here, we describe a simple chemical treatment with glutaraldehyde to enhance autofluorescence for 3D fluorescence imaging and to generate detailed morphological images on whole-mount samples. This methodology is straightforward and cost-effective to implement, suitable for a wide range of organisms and sample types. Furthermore, it can be readily integrated with standard confocal and fluorescence microscopes for analysis. This approach has the potential to facilitate the analysis of biological 3D structures and research in developmental biology, including studies on model and non-model organisms.

Joint subarray acoustic tweezers enable controllable cell translation, rotation, and deformation

Contactless microscale tweezers are highly effective tools for manipulating, patterning, and assembling bioparticles. However, current tweezers are limited in their ability to comprehensively manipulate bioparticles, providing only partial control over the six fundamental motions (three translational and three rotational motions). This study presents a joint subarray acoustic tweezers platform that leverages acoustic radiation force and viscous torque to control the six fundamental motions of single bioparticles. This breakthrough is significant as our manipulation mechanism allows for controlling the three translational and three rotational motions of single cells, as well as enabling complex manipulation that combines controlled translational and rotational motions. Moreover, our tweezers can gradually increase the load on an acoustically trapped cell to achieve controllable cell deformation critical for characterizing cell mechanical properties. Furthermore, our platform allows for three-dimensional (3D) imaging of bioparticles without using complex confocal microscopy by rotating bioparticles with acoustic tweezers and taking images of each orientation using a standard microscope. With these capabilities, we anticipate the JSAT platform to play a pivotal role in various applications, including 3D imaging, tissue engineering, disease diagnostics, and drug testing.

SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue.

Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-μm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72 h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.

Genetically-encoded markers for confocal visualization of single dense core vesicles.

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids.

The goal of this study was to establish a simple model of 3D endothelial spheroids with mosaic gene expression using adeno-associated virus (AAV) transduction, with a future aim being to study the activity of post-zygotic mutations common to vascular malformations. In this study, 96-well U-bottom plates coated with a commercial repellent were seeded with two immortalized human endothelial cell lines and aggregation monitored using standard microscopy or live-cell analysis. The eGFP expression was used to monitor the AAV transduction. HUVEC-TERT2 could not form spheroids spontaneously. The inclusion of collagen I in the growth medium could stimulate cell aggregation; however, these spheroids were not stable. In contrast, the hCMEC/D3 cells aggregated spontaneously and formed reproducible, robust 3D spheroids within 3 days, growing steadily for at least 4 weeks without the need for media refreshment. The hCMEC/D3 spheroids spontaneously developed a basement membrane, including collagen I, and expressed endothelial-specific CD31 at the spheroid surface. Serotypes AAV1 and AAV2QUADYF transduced these spheroids without toxicity and established sustained, mosaic eGFP expression. In the future, this simple approach to endothelial spheroid formation combined with live-cell imaging could be used to rapidly assess the 3D phenotypes and drug and radiation sensitivities arising from mosaic mutations common to brain vascular malformations.

An optical system for cellular mechanostimulation in 3D hydrogels

We introduce a method utilizing single laser-generated cavitation bubbles to stimulate cellular mechanotransduction in dermal fibroblasts embedded within 3D hydrogels. We demonstrate that fibroblasts embedded in either amorphous or fibrillar hydrogels engage in Ca2+ signaling following exposure to an impulsive mechanical stimulus provided by a single 250 µm diameter laser-generated cavitation bubble. We find that the spatial extent of the cellular signaling is larger for cells embedded within a fibrous collagen hydrogel as compared to those embedded within an amorphous polyvinyl alcohol polymer (SLO-PVA) hydrogel. Additionally, for fibroblasts embedded in collagen, we find an increased range of cellular mechanosensitivity for cells that are polarized relative to the radial axis as compared to the circumferential axis. By contrast, fibroblasts embedded within SLO-PVA did not display orientation-dependent mechanosensitivity. Fibroblasts embedded in hydrogels and cultured in calcium-free media did not show cavitation-induced mechanotransduction; implicating calcium signaling based on transmembrane Ca2+ transport. This study demonstrates the utility of single laser-generated cavitation bubbles to provide local non-invasive impulsive mechanical stimuli within 3D hydrogel tissue models with concurrent imaging using optical microscopy. Statement of significanceCurrently, there are limited methods for the non-invasive real-time assessment of cellular sensitivity to mechanical stimuli within 3D tissue scaffolds. We describe an original approach that utilizes a pulsed laser microbeam within a standard laser scanning microscope system to generate single cavitation bubbles to provide impulsive mechanostimulation to cells within 3D fibrillar and amorphous hydrogels. Using this technique, we measure the cellular mechanosensitivity of primary human dermal fibroblasts embedded in amorphous and fibrillar hydrogels, thereby providing a useful method to examine cellular mechanotransduction in 3D biomaterials. Moreover, the implementation of our method within a standard optical microscope makes it suitable for broad adoption by cellular mechanotransduction researchers and opens the possibility of high-throughput evaluation of biomaterials with respect to cellular mechanosignaling.

Unveiling the apoptotic potential of antioxidant-rich Bangladeshi medicinal plant extractives and computational modeling to identify antitumor compounds

Nowadays, there has been a significant surge in the exploration of anticancer compounds derived from medicinal plants due to their perceived safety and efficacy. Therefore, our objective was to investigate the antioxidant and antiproliferative properties, along with the phytoconstituents, of methanol extracts from various parts of 15 selected Bangladeshi medicinal plants. Standard spectrophotometric methods and confocal microscopy were utilized to assess the antioxidant and antiproliferative potential of these extracts. Additionally, phytochemical profiling was executed through gas chromatography-mass spectrometry (GC-MS) analysis. Among the extractives, Bombax ceiba bark exhibited the highest scavenging capacity against DPPH (IC50: 10.3 ± 0.7 μg/mL) and hydroxyl (IC50: 3.9 ± 0.1 μg/mL) free radicals. Furthermore, the total antioxidants, reducing power, and polyphenols of B. ceiba bark were higher than those of other extracts. B. ceiba bark also showed significant antiproliferative capacity against MCF-7 cells (86.67 %) in the MTT assay, followed by Cocos nucifera roots (83.92 %), Bixa orellana leaves (44.09 %), and Leea macrophylla roots (25 %). Moreover, B. ceiba bark, L. macrophylla roots, C. nucifera roots, and B. orellana leaves-treated Ehrlich ascites carcinoma (EAC) cells demonstrated growth inhibition rates of 87.27 %, 80.45 %, 42.9 %, and 37.27 %, respectively. Fluorescence microscopic analysis of EAC cells treated with these extracts revealed apoptotic features such as condensed chromatin, cell shrinkage, nucleus fragmentation, and membrane blebbing compared to untreated EAC cells. The GC-MS analysis of B. ceiba bark identified 18 compounds, including various alcohols, alkenes, and esters. Additionally, a molecular docking study revealed oxalic acid, cyclohexyl dodecyl ester as the most potent compound (−6.5) active against breast cancer. In summary, our results demonstrate that B. ceiba bark possesses robust antioxidant and antiproliferative properties, along with potent antitumor compounds, which could be utilized in the treatment of carcinoma.

SCIP: A scalable, reproducible, and open-source pipeline for morphological profiling image cytometry and microscopy data.

Imaging flow cytometry (IFC) provides single-cell imaging data at a high acquisition rate. It is increasingly used in image-based profiling experiments consisting of hundreds of thousands of multi-channel images of cells. Currently available software solutions for processing microscopy data can provide good results in downstream analysis, but are limited in efficiency and scalability, and often ill-adapted to IFC data. In this work, we propose Scalable Cytometry Image Processing (SCIP), a Python software that efficiently processes images from IFC and standard microscopy datasets. We also propose a file format for efficiently storing IFC data. We showcase our contributions on two large-scale microscopy and one IFC datasets, all of which are publicly available. Our results show that SCIP can extract the same kind of information as other tools, in a much shorter time and in a more scalable manner.

HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging.

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by Hoechst signal across the nucleus. We show a dose dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Design of an FPGA-Based Controller for Fast Scanning Probe Microscopy.

Atomic-scale imaging using scanning probe microscopy is a pivotal method for investigating the morphology and physico-chemical properties of nanostructured surfaces. Time resolution represents a significant limitation of this technique, as typical image acquisition times are on the order of several seconds or even a few minutes, while dynamic processes-such as surface restructuring or particle sintering, to be observed upon external stimuli such as changes in gas atmosphere or electrochemical potential-often occur within timescales shorter than a second. In this article, we present a fully redesigned field programmable gate array (FPGA)-based instrument that can be integrated into most commercially available standard scanning probe microscopes. This instrument not only significantly accelerates the acquisition of atomic-scale images by orders of magnitude but also enables the tracking of moving features such as adatoms, vacancies, or clusters across the surface ("atom tracking") due to the parallel execution of sophisticated control and acquisition algorithms and the fast exchange of data with an external processor. Each of these measurement modes requires a complex series of operations within the FPGA that are explained in detail.

Optical detection and enumeration of Escherichia coli and Salmonella enterica using a low-magnification light microscope

A rapid and cost-effective method for detecting bacterial cells from surfaces is critical to food safety, clinical hygiene, and pharmacy quality. Herein, we established an optical detection method based on a gold chip coating with 3-mercaptophenylboronic acid (3-MPBA) to capture bacterial cells, which allows for the detection and quantification of bacterial cells with a standard light microscope under low-magnification (10×) objective lens. Then, integrate the developed optical detection method with swab sampling to detect bacterial cells loading on stainless-steel surfaces. Using Salmonella enterica (SE1045) and Escherichia coli (E. coli OP50) as model bacterial cells, we achieved a capture efficiency of up to 76.0 ± 2.0 % for SE1045 cells and 81.1 ± 3.3 % for E. coli OP50 cells at 103 CFU/mL upon the optimized conditions, which slightly decreased with the increasing bacterial concentrations. Our assay showed good linear relationships between the concentrations of bacterial cells with the cell counting in images in the range of 103 -107 CFU/mL for SE1045, and 103 -108 CFU/mL for E. coli OP50 cells. The limit of detection (LOD) was 103 CFU/mL for both SE1045 and E. coli OP50 cells. A further increase in sensitivity in detecting E. coli OP50 cells was achieved through a heat treatment, enabling the LOD to be reduced as low as 102 CFU/mL. Furthermore, a preliminary application succeeded in assessing bacterial contamination on stainless-steel surfaces following integration with the approximately 40 % recovery rate, suggesting prospects for evaluating the bacteria from surfaces. The entire process was completed within around 2 h, costing merely a few dollars per sample. Considering the low cost of standard light microscopes, our method holds significant potential for practical industrial applications in bacterial contamination control on surfaces, especially in low-resource settings.

Clinical Profile and Outcomes of Patients With Systemic Lupus Erythematosus.

Background Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by relapsing-remitting immune system activation, affecting multiple organ systems. Despite significant advances in understanding SLE's pathogenesis, there remains a need for comprehensive clinical profiling at the time of diagnosis to improve early detection and management. This study addresses this gap by providing a detailed analysis of the clinical presentation, disease activity, and patient outcomes using the Systemic Lupus International Collaborating Clinics (SLICC) criteria and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) index. Methodology This cross-sectional observational study included 80 patients diagnosed with SLEusing the 2012 SLICC criteria. Patients were recruited from the Rheumatology department and other wards of Byramjee JeejeebhoyGovernment Medical Collegeand Sassoon General Hospital, Pune, India. All participants provided informed consent and institutional ethical approval was obtained. Data were collected through detailed clinical history, physical examinations, and standard tests such aschest X-rays, CBC, RFT, LFT, urine microscopy, creatine phosphokinase, ANA, AntiDsDNA, complement consumption, and Coombs' tests, with 2D echocardiography performed as needed. Follow-ups every three months over 1.5 years assessed disease activity using SLEDAI criteria. Patients aged 12 and above who met the SLICC criteria were included and those with other connective tissue disorders were excluded. Associations between clinical symptoms and organ involvement were analyzed using thechi-square testwith a p-value of <0.05 considered significant. Results The study evaluated 80 patients with SLE, revealing a predominantly female cohort (80%) with a mean age of 29.4 years and a standard deviation of 8.3 years, skewed towards younger age groups. Clinical manifestations were diverse; the most common symptoms were (83.75%), oral ulcers (98.75%), and alopecia (95%). Anemia (66.25%) was the most prevalent abnormality, followed by albuminuria and renal abnormalities. Organ involvement was highest in the renal system (50%) and mucocutaneous features, with lower incidences in cardiac, gastrointestinal, and vascular systems. Gender-specific analyses indicated significant differences in SLE nephritis (p=0.048) and autoimmune hemolytic anemia (p=0.046). Autoantibody profiles showed high positivity for ANA (98.8%) and DsDNA (61.3%). Clinical outcomes demonstrated that 68.8% of patients achieved remission and 16.3% experienced organ damage. The SLEDAI scores significantly improved over time, with substantial reductions from baseline to nine months (p<0.001). Conclusion In conclusion, this study provides a detailed examination of SLE, revealing that it predominantly affects young adults and is characterized by diverse manifestations including mucocutaneous symptoms, significant renal involvement, and notable autoantibody profiles. The high prevalence of anti-nucleosome and anti-dsDNA antibodies underscores their diagnostic and prognostic value. Clinically, the findings highlight the necessity for early detection and targeted management of SLE, particularly in addressing renal and mucocutaneous symptoms. Future research should focus on longitudinal studies to track disease progression, explore genetic and environmental influences, and investigate regional variations to enhance treatment strategies and patient outcomes.

Leaf micromorphology and anatomical traits of leaves as potential taxonomic markers for infrageneric classification of&lt;em&gt; Cinnamomum&lt;/em&gt; (Lauraceae)

This study aimed to examine the leaf anatomy of the eight species of Cinnamomum (family Lauraceae) that occur in Sri Lanka to evaluate its potential utility in taxonomic delimitation. Leaf surface micromorphology and internal anatomy were studied using standard light and scanning electron microscopy. Infrageneric and interspecific variation in micromorphology of cuticular materials was observed in both abaxial and adaxial surfaces. Both abaxial and adaxial surfaces had undulating, thick cuticles with different patterns of wax deposition. Leaves were hypostomatic with no special arrangement for epidermal pavement cells. Simple, unicellular, unbranched, solitary and non-glandular trichomes were observed in different species on both surfaces of the leaves. Both adaxial and abaxial surfaces in C. dubium and C. capparu-coronde were densely covered with simple, unicellular/ falcate, long and thin trichomes. Transverse sections (TS) of the leaves were different in shape (symmetrical, asymmetrical, irregular, saucer). The midrib contained one open arch central vascular bundle that was different in shape (oval, elongated, irregular, ‘V’shaped, partially dissected into 2 or 3 segments) in different species. The number of collateral vessels was dissimilar between the species and varied in number (±10 - ±17). Isolated or aggregated crystals were observed in different parts of the leaf and oval-to-round schizogenous secretory cells were present in C. capparu-coronde, C. citriodorum and C. dubium. Leaf cuticular features together with the presence/absence of secretory cavities, trichome shape and density, midrib cross-section outline and shape of vascular bundles are taxonomically informative characteristics that can be used to differentiate the eight species of Cinnamomum that occur in Sri Lanka.

Direct electron beam writing of silver using a β-diketonate precursor: first insights.

Direct electron beam writing is a powerful tool for fabricating complex nanostructures in a single step. The electron beam locally cleaves the molecules of an adsorbed gaseous precursor to form a deposit, similar to 3D printing but without the need for a resist or development step. Here, we employ for the first time a silver β-diketonate precursor for focused electron beam-induced deposition (FEBID). The used compound (hfac)AgPMe3 operates at an evaporation temperature of 70-80 °C and is compatible with commercially available gas injection systems used in any standard scanning electron microscope. Growth of smooth 3D geometries could be demonstrated for tightly focused electron beams, albeit with low silver content in the deposit volume. The electron beam-induced deposition proved sensitive to the irradiation conditions, leading to varying compositions of the deposit and internal inhomogeneities such as the formation of a layered structure consisting of a pure silver layer at the interface to the substrate covered by a deposit layer with low silver content. Imaging after the deposition process revealed morphological changes such as the growth of silver particles on the surface. While these effects complicate the application for 3D printing, the unique deposit structure with a thin, compact silver film beneath the deposit body is interesting from a fundamental point of view and may offer additional opportunities for applications.

One step 4x and 12x 3D-ExM: robust super-resolution microscopy in cell biology.

Super-resolution microscopy has become an indispensable tool across diverse research fields, offering unprecedented insights into biological architectures with nanometer scale resolution. Compared to traditional nanometer-scale imaging methods such as electron microscopy, super-resolution microscopy offers several advantages, including the simultaneous labeling of multiple target biomolecules with high specificity and simpler sample preparation, making it accessible to most researchers. In this study, we introduce two optimized methods of super-resolution imaging: 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (4x and 12x 3D-ExM). 3D-ExM is a straightforward expansion microscopy method featuring a single-step process, providing robust and reproducible 3D isotropic expansion for both 2D and 3D cell culture models. With standard confocal microscopy, 12x 3D-ExM achieves a lateral resolution of under 30 nm, enabling the visualization of nanoscale structures, including chromosomes, kinetochores, nuclear pore complexes, and Epstein-Barr virus particles. These results demonstrate that 3D-ExM provides cost-effective and user-friendly super-resolution microscopy, making it highly suitable for a wide range of cell biology research, including studies on cellular and chromatin architectures.

OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images.

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

Loop-Mediated Isothermal Amplification for Diagnosis of Zoonotic Malaria.

Zoonotic malaria, caused by Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium coatneyi, and Plasmodium inui, is a significant global health concern. The gold standard microscopy, while widely used for malaria diagnosis, faces limitations in differentiating between malaria species. Polymerase chain reaction (PCR), despite its accuracy, is characterized by high costs and time-consuming procedures. This study aims to develop and validate a rapid and accurate diagnostic test for detecting four simian Plasmodium species by using loop-mediated isothermal amplification (LAMP). Loop-mediated isothermal amplification is a cost-effective and faster molecular testing alternative for malaria diagnosis. The project involved designing specific primers, testing sensitivity and specificity against various parasites (including human Plasmodium species, protozoa, and helminths), and evaluating the LAMP assay using 60 macaque samples infected with simian Plasmodium. The LAMP assay exhibited a sensitivity profile enabling the detection of P. knowlesi, P. coatneyi, and P. cynomolgi across a concentration gradient from 5 × 108 down to 5 × 105 parasites/µL. Notably, P. inui was detectable at 5 × 108 parasites/µL. Furthermore, the specificity of the primer tailored for the four simian Plasmodium species was proven, as it produced a positive amplification exclusively for the respective target species and generated negative results for nontarget species. The results indicated that the LAMP assay is capable of detecting simian Plasmodium within a short span of 60 minutes, without any false positives from other samples. This new test has the potential to revolutionize malaria diagnosis, surveillance, and control, thereby mitigating the impact of zoonotic malaria in regions of endemicity.

Morphological changes and luminescence of Escherichia coli in contact with Mn2O3 and Co3O4 ultrafine particles as components of a mineral feed additive.

The spread of antibiotic resistance and mineral depletion in soils encourages an intensive search for highly effective and environmentally safe bactericidal agents and sources of macro- and micro-elements. The most profitable solution would combine both the described tasks. Ultrafine particles (UFPs) have this functionality. Thus, this study aimed to analyze the bioluminescence and external morphological changes of Escherichia coli cells after contact with M2O3 and Co3O4 UFPs at effective concentrations (ECs). The antibiotic properties of the studied samples were determined on a multifunctional microplate analyzer TECAN Infinite F200 (Tecan Austria GmbH, Austria) by fixing the luminescence value of the bacterial strain E. coli K12 TG11 (Ecolum, NVO Immunotech Closed Joint Stock Company, Russia). Morphological changes in the cell structure were evaluated using a Certus Standard EG-5000 atomic force microscope equipped with NSPEC software (Nano Scan Technology LLC, Russia). The obtained results indicate high bactericidal properties of Co3O4 and Mn2O3 UFPs (EC50 at 3.1 × 10-5 and 1.9 × 10-3 mol/L, respectively) due to the degradation of the cell wall, pathological increase in size, disruption of septic processes, and loss of cytoplasmic contents. The prospects for the environmentally safe use of ultrafine materials are outlined. The limits of the dosages of Co3O4 and Mn2O3 UFPs recommended for further study in vitro and in vivo in feeding farm animals are established (no more than 4.9 × 10-4 mol/L for Mn2O3 UFPs and 1.5 × 10-5 mol/L for Co3O4 UFPs). The limitation of the work is the lack of experiments to determine the mechanisms of the toxic effect of UFP on bacteria, protein structures, and DNA and oxidative stress, which is planned to be performed in the future together with in situ and in vivo studies on animals.

Complementary testing and machine learning techniques for the characterization and prediction of middle Permian tight gas sandstone reservoir quality in the northeastern Ordos Basin, China

In this study, an integrated approach for diagenetic facies classification, reservoir quality analysis and quantitative wireline log prediction of tight gas sandstones (TGSs) is introduced utilizing a combination of fit-for-purpose complementary testing and machine learning techniques. The integrated approach is specialized for the middle Permian Shihezi Formation TGSs in the northeastern Ordos Basin, where operators often face significant drilling uncertainty and increased exploration risks due to low porosities and micro-Darcy range permeabilities. In this study, detrital compositions and diagenetic minerals and their pore type assemblages were analyzed using optical light microscopy, cathodoluminescence, standard scanning electron microscopy, and X-ray diffraction. Different types of diagenetic facies were delineated on this basis to capture the characteristic rock properties of the TGSs in the target formation. A combination of He porosity and permeability measurements, mercury intrusion capillary pressure and nuclear magnetic resonance data was used to analyze the mechanism of heterogeneous TGS reservoirs. We found that the type, size and proportion of pores considerably varied between diagenetic facies due to differences in the initial depositional attributes and subsequent diagenetic alterations; these differences affected the size, distribution and connectivity of the pore network and varied the reservoir quality. Five types of diagenetic facies were classified: (ⅰ) grain-coating facies, which have minimal ductile grains, chlorite coatings that inhibit quartz overgrowths, large intergranular pores that dominate the pore network, the best pore structure and the greatest reservoir quality; (ⅱ) quartz-cemented facies, which exhibit strong quartz overgrowths, intergranular porosity and a pore size decrease, resulting in the deterioration of the pore structure and reservoir quality; (ⅲ) mixed-cemented facies, in which the cementation of various authigenic minerals increases the micropores, resulting in a poor pore structure and reservoir quality; (ⅳ) carbonate-cemented facies and (ⅴ) tightly compacted facies, in which the intergranular pores are filled with carbonate cement and ductile grains; thus, the pore network mainly consists of micropores with small pore throat sizes, and the pore structure and reservoir quality are the worst. The grain-coating facies with the best reservoir properties are more likely to have high gas productivity and are the primary targets for exploration and development. The diagenetic facies were then translated into wireline log expressions (conventional and NMR logging). Finally, a wireline log quantitative prediction model of TGSs using convolutional neural network machine learning algorithms was established to successfully classify the different diagenetic facies.

Use of Color Channel Optimization in 3D Heads-Up Vitrectomy vs Standard Operating Microscope for Macular Surgeries.

Purpose: To compare the efficacy and safety of color channel optimization with 3-dimensional (3D) heads-up vitrectomy (3D HUD group) vs standard operating microscope vitrectomy (control group) for macular surgery. Methods: This retrospective multicenter comparative study comprised patients having 25-gauge pars plana vitrectomy for macular hole, epiretinal membrane (ERM), or vitreomacular traction. The minimum follow-up was 6 months. Surgeons completed a subjective questionnaire after each case. The main outcome measures were safety related (dye reinjection rate, macular ERM or internal limiting membrane [ILM] peeling time, endoillumination intensity). Other outcome measures included total surgical time, surgical outcomes, and subjective surgeon-related parameters. Results: The study included 74 eyes (36 in 3D HUD group; 38 in control group). There were no statistical differences in baseline parameters between groups. Significantly more eyes in the control group than in the 3D HUD group required dye reinjection (23.7% vs 5.6%; P = .03). Less time was required for ERM and ILM peeling in the 3D HUD group (both P < .01); however, the total surgical time was the same between groups. Eyes in the 3D HUD group required lower endoillumination (P < .001). There were no between-group differences in the rates of complications. Surgeons said depth perception was better in the control group (P < .001), with no differences in comfort or visibility. Conclusions: 3D heads-up-based color channel optimization for macular surgeries is safe and effective. Although it may have safety advantages, it did not affect the visual or anatomic outcomes or total surgical time and did not improve surgeon comfort or visibility.