Digital Micrographs Research Articles

Automatic cell identification and counting of leaf epidermis for plant phenotyping

The counting of leaf epidermal cells is useful to study the plant developmental changes produced by environmental or genetic factors.The scanning electron microscopy can be used, but it is expensive and time-consuming. Methods using optical microscopy are also available, but they still require leaves pre-treatment and manual cell identification.We propose a quick and simple method for counting leaf epidermal cells without leaf treatments and based on automated cell identification and marking. It allows to highly improve the representativeness of leaf epidermis screening, aiming at a high-throughput plant phenotyping approach.•The leaves are pressed between two glass slides without any pre-treatment and digital micrographs are acquired under incident light.•Epidermal cells are automatically identified and counted by means of a “macro" of ImageJ•The cell count obtained applying the procedure of image processing is very close to that obtainable by manual cell identification.

The use of convolutional neural networks to identify artifacts of cells micrographs in biomedical research

The work is devoted to the use of artificial neural networks to solve the problem of recognition and isolation of objects (cells) in digital micrographs used in the practice of microbiological research. This task is relevant due to a combination of two factors: a large amount of data (hundreds and thousands of photographs, hundreds of objects on each) arising from such studies, as well as the high complexity of manual image processing and the risk of operator errors. To solve the problem of recognition and separation of objects (cells) in digital biomedical micrographs, artificial convolutional neural networks are used. However, in most cases, the use of artificial neural networks requires selection of the network architecture and its training for each task individually. Each time when solving the problem of training the network, it is necessary to collect huge training samples, the more complex the more complex the architecture. The task of cell isolation was solved using a convolutional neural network. The neural network architecture of Mask R-CNN Facebook Research was used. We used a pretrained neural network, retrained in digital micrographs obtained and marked out by the authors. The solution is implemented in Python using TensorFlow, an open source machine learning software library developed by Google. As a result of testing the system, on the available data, the correctness of cell recognition in microphotographs was more than 95%, despite the small size of the digital images used. The applied approach definitely showed its efficiency on the available experimental data and has development prospects in the direction of increasing the size of processed images, increasing recognition accuracy, expanding the composition of secreted objects, working not only with cells, but also with structures in tissues.

Formation of Stem Cell Aggregates and Their Differentiation on Surface-Patterned Hydrogels Based on Poly(2-hydroxyethyl Methacrylate).

Micropillar patterns were fabricated and used to study cell adhesion, morphology, and function. Micropillars were produced in poly(2-hydroxyethyl methacrylate (HEMA)/N,N-(dimethylaminoethyl)methacrylate (DMAEMA)/tetraethylene glycol dimethacrylate (TEGDMA)) hydrogels using soft lithography, had dimensions of 1 μm diameter, and were either 2.05 or 4.91 μm tall. The patterned hydrogel substrates increased adhesion and induced the formation of cellular aggregates. Digital micrographs were used to quantify aggregate size and number. Differentiation of hMSCs toward adipocytes and chondrocytes was performed using the respective complete culture and differentiation medium for 2 weeks. Cells were stained for Oil red O, Alcian blue, and Type II collagen. Hydrogel substrates supported the differentiation of hMSCs to adipocytes and chondrocytes. The taller micropillar patterns supported the attachment and growth of larger aggregates and were more amenable to aid chondrogenic differentiation.

SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets—typically comprising thousands of particles—is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200–2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.

Uticaj litijum sulfata na Šenonovu entropiju hromatina kod limfocita

Introduction: Lithium affects numerous signal pathways in cells, which may ultimately lead to either increased or decreased gene expression in cell nuclei. However, effects of lithium on higher level of gene organization in the nuclei, like chromatin, is still poorly understood. Aim: To investigate the effects of lithium on chromatin organization in the nuclei of lymphocytes by investigating changes in Shannon entropy of chromatin in these cells. Material and Methods: Peripheral blood was treated with lithium sulphate until lithium concentrations of 1mmol/l, 2 mmol/l i 3 mmol/l was not reached. The smears were fixed with methanol and stained by Felgen method for DNK visualization. Cells in the smear were treated with hydrochloric acid for 120 min. Schiff reagent was used for staining for 120 min and smear washing was done with 6 ml of 10% of water solution of natrium metabisulfite. After staining, digital micrographs for each of the most representative sample of 30 lymphocytic chromatin structures were made. Shannon entropy was calculated by converting micrograph in the format of textual numerical string. Lymphocyte chromatin was also analyzed by textural method analysis. Results: Treatment with 1 mmol/l of lithium sulphate did not lead to a statistically significant increase in entropy values (p > 0.05). However, in samples where the concentration of lithium was 2 i 3 mmol/l, respectively, there was a statistically significant increase in entropy (p < 0.05). Also, a statistically significant and dose-dependent linear trend of an increase of chromatin entropy was detected in samples (p < 0.05). After lithium sulphate treatment, neither the mean value of angular second moment lymphocyte chromatin in control sample nor the inverse moment of the difference of treated lymphocytes changed (p > 0.05). Conclusion: Lithium sulphate in peripheral blood lymphocytes causes dose-dependent increase in Shannon chromatin entropy, which is not followed with similar changes in a textural chromatin parameters.

Iron oxide nanoparticles decrease nuclear fractal dimension of buccal epithelial cells in a time-dependent manner.

In this paper, we present results that iron oxide nanoparticles (IONPs) induce time-dependent structural changes in nuclei of buccal epithelial cells. The cells were treated with magnetite, Fe3 O4 nanoparticles (spherical shape, diameter 80-100 nanometres). The digital micrographs of the nuclei were made in 3 different time points: 15, 30 and 60 min after the treatment with IONPs, as well as in the control cells. A total of 120 nuclear structures (30 per sample) were analysed. Fractal analysis of nuclei was done in ImageJ software of the National Institutes of Health, (Bethesda, MD, USA). For each nuclear structure, the values of fractal dimension and lacunarity were calculated. There was a time-dependent reduction of nuclear fractal dimension in buccal epithelial cells after exposure to magnetite iron oxide nanoparticles. Negative trend was observed (p<0.01). Nuclear lacunarity, as another fractal parameter was shown to increase, also in a time-dependent manner, after the treatment with IONPs. To our knowledge, this is the first study to investigate effects of magnetite nanomaterial on nuclear fractal complexity, and also the first to apply fractal analysis method in testing of the interaction between nanoparticles and cell nucleus in this experimental setting.

Coarsening of Inter- and Intra-granular Proeutectoid Cementite in an Initially Pearlitic 2C-4Cr Ultrahigh Carbon Steel

We have examined spheroidization and coarsening of cementite in an initially pearlitic 2C-4Cr ultrahigh carbon steel containing a cementite network. Coarsening kinetics of spheroidized cementite and growth of denuded zones adjacent to the cementite network were investigated by analyzing particle sizes from digital micrographs of water-quenched steel etched with Nital. Denuded zones grew at a rate proportional to t 1/4–t 1/5. Spheroidization of pearlite was completed within 90 minutes at 1073 K and 1173 K (800 °C and 900 °C), and within 5 minutes at 1243 K (970 °C). Bimodal particle size distributions were identified in most of the samples and were more pronounced at higher temperatures and hold times. Peaks in the distributions were attributed to the coarsening of intragranular and grain boundary particles at different rates. A third, non-coarsening peak of particles was present at 1073 K (800 °C) only and was attributed to particles existing prior to the heat treatment. Particle sizes were plotted vs time to investigate possible coarsening mechanisms. The coarsening exponent for the growth of grain boundary carbides was closest to 4, indicating grain boundary diffusion control. The coarsening exponent was closest to 5 for intragranular carbides, indicating suppression of volumetric diffusion (possibly due to reduced effective diffusivity because of Cr alloying) and control by dislocation diffusion.

Multi-fractal morphology of un-aged and aged SBS polymer-modified bitumen

ABSTRACTPolymer-modified bitumen (PmBs) has been characterised by fluorescence microscopy, conventional performance tests and by the rheological measurements, before and after the Rolling Thin Film Oven Test (RTFOT), and by multi-fractal analysis. The addition of 2–7 wt-% of linear styrene-butadiene-styrene block copolymer (SBS-L) or radial block copolymer (SBS-R) increased viscosity, softening point and complex modulus of PmBs. Hardening of PmBs occurred and the elastic response decreased after RTFOT aging. SBS-L PmBs are more susceptible to RTFOT aging. Digital fluorescence micrographs of sections of PmBs qualitatively exhibit different morphological structures which can be quantitatively probed by multi-fractal analysis: differences are reflected in the f (α) spectrum. The PmBs which appear less multi-fractal have the best rheological properties. In another approach, the focal fractal dimensions’ distributions for the SBS-modified bitumen have peak around 2.35 (which corresponds to the bitumen-rich phase) and around 2.7 (which corresponds to the noncontinuous SBS-rich phase).

REDESCRIPTION AND NEW DATA ON THE DISTRIBUTION OF A POORLY KNOWN PIRATE SPIDER ERMETUS INOPINABILIS (ARANEI: MIMETIDAE)

&lt;p&gt;A poorly known species of pirate spider belonging to a monotypic genus, &lt;em&gt;Ermetus inopinabilis &lt;/em&gt;Ponomarev, 2008, is redescribed in details based on types and other material. Digital photographs and SEM micrographs are provided to illustrate its specific features. The species and genus are reported for the first time from Altai and Tuva.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;

Additive Manufacturing of Single-Crystal Superalloy CMSX-4 Through Scanning Laser Epitaxy: Computational Modeling, Experimental Process Development, and Process Parameter Optimization

This paper focuses on additive manufacturing (AM) of single-crystal (SX) nickel-based superalloy CMSX-4 through scanning laser epitaxy (SLE). SLE, a powder bed fusion-based AM process was explored for the purpose of producing crack-free, dense deposits of CMSX-4 on top of similar chemistry investment-cast substrates. Optical microscopy and scanning electron microscopy (SEM) investigations revealed the presence of dendritic microstructures that consisted of fine γ′ precipitates within the γ matrix in the deposit region. Computational fluid dynamics (CFD)-based process modeling, statistical design of experiments (DoE), and microstructural characterization techniques were combined to produce metallurgically bonded single-crystal deposits of more than 500 μm height in a single pass along the entire length of the substrate. A customized quantitative metallography based image analysis technique was employed for automatic extraction of various deposit quality metrics from the digital cross-sectional micrographs. The processing parameters were varied, and optimal processing windows were identified to obtain good quality deposits. The results reported here represent one of the few successes obtained in producing single-crystal epitaxial deposits through a powder bed fusion-based metal AM process and thus demonstrate the potential of SLE to repair and manufacture single-crystal hot section components of gas turbine systems from nickel-based superalloy powders.

Small Bugs, Big Changes: Taxonomic Revision of Orthorhagus McAtee & Malloch.

The Schizopteridae, the largest family of litter bugs (Hemiptera: Schizopteridae), comprises 53 genera of primarily tropical, tiny true bugs. The largest genus Schizoptera Fieber comprises 64 described species and six subgenera with primarily tropical and subtropical distributions across the New World. Schizoptera species are morphologically diverse with extreme asymmetry of male genitalia and sexually dimorphic wing in females of some species. Subgeneric concepts of Schizoptera are yet to be evaluated in a phylogenetic context. The subgenus Orthorhagus McAtee & Malloch was described from a single specimen as part of Schizoptera based on similarities of wing venation and other structures. The absence of a pronotal collar was used to separate Orthorhagus from other subgenera within Schizoptera, but morphological and molecular data show that the differences between Orthorhagus species and those of Schizoptera extend beyond the lack of a pronotal collar. In this study, we elevate the subgenus Orthorhagus to generic rank and describe five new species in this genus. We provide morphological documentation including digital habitus images, genitalic drawings, and confocal micrographs for all species. Distribution maps and a key to the species of Orthorhagus, n. stat. are also presented.

First Report of Orchid fleck virus in Lilyturf (Liriope spicata) in Australia

Liriope spicata (Asparagaceae) is an evergreen ornamental plant commonly referred to as lilyturf, creeping liriope, monkey grass, or creeping lilyturf, and is widely grown as groundcover and for erosion control (Fantz 2008). In April 2015, lilyturf plants with bright yellow flecks on the leaves were observed in landscape plantings across the St. Lucia campus of the University of Queensland, Brisbane, Australia. These symptoms were reminiscent of those observed on leaves of orchid species infected by Orchid fleck virus (OFV) (Kubo et al. 2009). Symptomatic leaf samples were collected and analyzed in a JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan). Leaf dips were prepared by breaking up symptomatic tissue in 1% ammonium molybdate, pH 7.0 negative stain and extracts were placed on nitrocellulose-coated copper grids. Digital micrographs were captured with an Orius digital camera (Gatan Inc., Pleasanton, CA). Typical nonenveloped, rhabdovirus-like, bacilliform particles measuring ∼45 × 95 nm were observed, similar to those of OFV from infected orchids (Kondo et al. 2006). Total RNA was extracted from symptomatic tissue using an RNeasy Plant Mini Kit (Qiagen) and subjected to RT-PCR using the Superscript III One Step RT-PCR System with Platinum Taq DNA Polymerase (Thermo Fisher) and OFV nucleoprotein (N) gene primers polydT/SP6 (5′-GATTTAGGTGACACTATAGTTTTTTTTTTTTTTTTT(A/G/C)-3′) and mN2 (5′-TGCAGGAATATAGCCGACATGTT-3′) (Blanchfield et al. 2001). Agarose gel electrophoresis showed a single amplicon of ∼800 bp, comparable to the OFV positive control. DNA from both the lilyturf (DAF Plant Virus Collection Accession No. 5216) and a cymbidium OFV control (Accession No. 703) amplicons were gel-purified and cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced using dideoxynucleotide sequencing at the Australian Genome Research Facility (Brisbane, Australia). A GenBank BLASTx search of the lilyturf virus N gene sequence of 658 nt showed 100% match to the N protein sequence of an Australian OFV isolate (Genbank Accession No. AAK09392 and e-value 1e-143). The partial N gene nucleotide sequence of the lilyturf OFV isolate #5216 was 99% identical to the cymbidium OFV isolate. Both sequences have been deposited in GenBank with Accession Nos. KT947974 and KT947975, respectively. Based on the observed symptoms, particle morphology, N gene amplicon using OFV primers, and high sequence identity with other OFV sequences, we conclude that the lilyturf plants were infected with OFV. This is the first report of OFV infecting L. spicata. This alternative host may play a possible role in OFV spread to orchids or other ornamental species that are colonized by its Brevipalpus sp. mite vector.

Prognostic criteria of chronic myeloid leukemia by changes platelets sizes.

In order to identify specific changes in the size of platelets in patients with chronic myeloid leukemia (CML) and assess possibility of their use as prognostic criteria for disease progression peripheral blood of 62 patients with CML was examined. The study was carried out by an automatic hematology analyzer and method of computer image analysis on micrographs of peripheral blood smears. CML patients showed an increase in mean platelet volume (parametr MPV) and the percentage of large platelets (parameter P-LCR). Measurement of platelet digital micrographs, carried out in parallel, showed an increase of variability of their size. Analysis of characteristic curves of the specificity/sensitivity showed the possibility of using the coefficient of variation and standard deviation of the natural logarithm of platelets in areas of the peripheral blood smear as a predictor of progression of CML in blast crisis phase.

Fractal analysis and Gray level co-occurrence matrix method for evaluation of reperfusion injury in kidney medulla

Fractal analysis and Gray level co-occurrence matrix method represent two novel mathematical algorithms commonly used in medical sciences as potential parts of computer-aided diagnostic systems. In this study, we tested the ability of these methods to discriminate the kidney medullar tissue suffering from reperfusion injury, from normal tissue. A total of 320 digital micrographs of Periodic acid-Schiff (PAS) – stained kidney medulla from 16 Wistar albino mice (20 per animal), were analyzed using National Institutes of Health ImageJ software (NIH, Bethesda, MD) and its plugins. 160 micrographs were obtained from the experimental group with induced reperfusion injury, and another 160 were obtained from the controls. For each micrograph we calculated the values of fractal dimension, lacunarity, as well as five GLCM features: angular second moment, entropy, inverse difference moment, GLCM contrast, and GLCM correlation. Discriminatory value of the parameters was tested using receiver operating characteristic (ROC) analysis, by measuring the area below ROC curve. The results indicate that certain features of GLCM algorithm have excellent discriminatory ability in evaluation of damaged kidney tissue. Fractal dimension and lacunarity as parameters of fractal analysis also had a relatively good discriminatory value in differentiation of injured from the normal tissue. Both methods have potentially promising application in future design of novel techniques applicable in cell physiology, histology and pathology.

2016

The purpose of this study was to evaluate and compare the intrusion characteristics of Simplex-HV to Simplex-P and Palacos-R in cadaveric proximal tibial bone. Eighteen fresh-frozen cadaver proximal tibiae were examined with standard arthroplasty tibial cuts. Each tibia was randomly assigned to receive one of the three bone cements for use with finger packing technique. Sagittal sections were prepared and analyzed using digital photography and stereoscopic micrographs to evaluate cement intrusion characteristics. The cement penetration depth was measured from the tibial bone cut surface, which did not include the cement thickness under the tibial base plate. Significant differences were detected in the bone cement penetration between the three cements. Penetration was increased using the Simplex-HV (average, 2.7 mm; range, 2.0-3.0 mm) compared with Simplex-P (average, 2.2 mm) and Palacos-R (average, 1.8 mm). These depths approximate to 3.7, 3.2, and 2.8 mm of total cement penetration, respectively. The data suggest that high-viscosity bone cement may provide good fixation of the tibial component of a total knee arthroplasty when using the finger packing technique.

Quantification of structural changes in acute inflammation by fractal dimension, angular second moment and correlation.

The aim of the study was to examine alteration and possible application of fractal dimension, angular second moment, and correlation for quantification of structural changes in acutely inflamed tissue. Acute inflammation was induced by injection of turpentine oil into the right and left hind limb muscles of mice, whereas control animals received intramuscular saline injection. After 12 h, animals were anesthetised and treated muscles collected. The tissue was stained by hematoxylin and eosin, digital micrographs produced, enabling determination of fractal dimension of the cells, angular second moment and correlation of studied tissue. Histopathological analysis showed presence of inflammatory infiltrate and tissue damage in inflammatory group, whereas tissue structure in control group was preserved, devoid of inflammatory infiltrate. Fractal dimension of the cells, angular second moment and correlation of treated tissue in inflammatory group decreased in comparison to the control group. In this study, we were first to observe and report that fractal dimension of the cells, angular second moment, and correlation were reduced in acutely inflamed tissue, indicating loss of overall complexity of the cells in the tissue, the tissue uniformity and structure regularity. Fractal dimension, angular second moment and correlation could be useful methods for quantification of structural changes in acute inflammation.

Methodology for the use of software for the analysis of digital micrographs on the base of pathomorphology course in order to increase the professional level of students and scientists.

Methodology for the use of software for the analysis of digital micrographs on the base of pathomorphology course in order to increase the professional level of students and scientists.

Correlations between body length and otolith size in smallmouth bass Micropterus dolomieu Lacépède, 1802 with implications for retrospective growth analyses

Reconstructing individual growth history from analysis of increments in otoliths, scales, or spines can provide information on past growth responses to environmental variation, which in turn can be useful for predicting population-level response to climate change. The objective of this study was to examine correlations between body length and different metrics of otolith size for Micropterus dolomieu. Three metrics corresponding to commonly-used microstructural and ultrastructural otolith dimensions were measured using image analysis of digital micrographs from a sample of 214 M. dolomieu ranging from 115 to 438 mm total length collected in 2011–2013. It was found that anteroposterior length of whole otoliths provided much improved regression relationships with body size as well as ease of data collection and faster sampling throughput compared with microstructural measures from polished sections. When applying these metrics to reconstruct growth history the biological intercept model generally produced more reasonable back-calculated estimates of length-at-age, although this was not consistent across all otolith metrics. Results suggest that whole otolith measures should be employed due to efficiency of data collection and greater reliability for reconstructing growth history in M. dolomieu.

Additive Manufacturing of IN100 Superalloy Through Scanning Laser Epitaxy for Turbine Engine Hot-Section Component Repair: Process Development, Modeling, Microstructural Characterization, and Process Control

This article describes additive manufacturing (AM) of IN100, a high gamma-prime nickel-based superalloy, through scanning laser epitaxy (SLE), aimed at the creation of thick deposits onto like-chemistry substrates for enabling repair of turbine engine hot-section components. SLE is a metal powder bed-based laser AM technology developed for nickel-base superalloys with equiaxed, directionally solidified, and single-crystal microstructural morphologies. Here, we combine process modeling, statistical design-of-experiments (DoE), and microstructural characterization to demonstrate fully metallurgically bonded, crack-free and dense deposits exceeding 1000 μm of SLE-processed IN100 powder onto IN100 cast substrates produced in a single pass. A combined thermal-fluid flow-solidification model of the SLE process compliments DoE-based process development. A customized quantitative metallography technique analyzes digital cross-sectional micrographs and extracts various microstructural parameters, enabling process model validation and process parameter optimization. Microindentation measurements show an increase in the hardness by 10 pct in the deposit region compared to the cast substrate due to microstructural refinement. The results illustrate one of the very few successes reported for the crack-free deposition of IN100, a notoriously “non-weldable” hot-section alloy, thus establishing the potential of SLE as an AM method suitable for hot-section component repair and for future new-make components in high gamma-prime containing crack-prone nickel-based superalloys.

Discriminatory ability of fractal and grey level co-occurrence matrix methods in structural analysis of hippocampus layers.

Fractal and grey level co-occurrence matrix (GLCM) analysis represent two mathematical computer-assisted algorithms that are today thought to be able to accurately detect and quantify changes in tissue architecture during various physiological and pathological processes. However, despite their numerous applications in histology and pathology, their sensitivity, specificity and validity regarding evaluation of brain tissue remain unclear. In this article we present the results indicating that certain parameters of fractal and GLCM analysis have high discriminatory ability in distinguishing two morphologically similar regions of rat hippocampus: stratum lacunosum-moleculare and stratum radiatum. Fractal and GLCM algorithms were performed on a total of 240 thionine-stained hippocampus micrographs of 12 male Wistar albino rats. 120 digital micrographs represented stratum lacunosum-moleculare, and another 120 stratum radiatum. For each image, 7 parameters were calculated: fractal dimension, lacunarity, GLCM angular second moment, GLCM contrast, inverse difference moment, GLCM correlation, and GLCM variance. GLCM variance (VAR) resulted in the largest area under the Receiver operating characteristic (ROC) curve of 0.96, demonstrating an outstanding discriminatory power in analysis of stratum lacunosum-moleculare (average VAR equaled 478.1 ± 179.8) and stratum radiatum (average VAR of 145.9 ± 59.2, p < 0.0001). For the criterion VAR ≤ 227.5, sensitivity and specificity were 90% and 86.7%, respectively. GLCM correlation as a parameter also produced large area under the ROC curve of 0.95. Our results are in accordance with the findings of our previous study regarding brain white mass fractal and textural analysis. GLCM algorithm as an image analysis method has potentially high applicability in structural analysis of brain tissue cytoarcitecture.