Digital Micrographs Research Articles

Специализированное программное обеспечение анализа оптической плотности цифровых изображений микропрепаратов

The development of software for densitometric analysis is actual nowadays. The main task in performing the densitometric analysis is the estimation of light quantity rate which passed through the product of histochemical reaction of examined object to some sample transparent field without an analyzed object, delaying the light flow, such as slide-glass, coverslip, mounts environment. As a a rule, the information on the color of reaction products is of no interest to the study. That is why monochrome digital cameras are preferred for densitometric analysis. The article presents the issues of creating software for the analysis of digital micrographs of biological objects. The introduction sets out the problem statement and artifacts that result in errors during the densitometric analysis. The problem of constructing software for digital image processing micropreparations obtained using a microscope. The realization of software, mathematical methods of treatment, the results of applying these methods to real data.

First record of the genus Tagalis Stål, 1860 (Hemiptera: Reduviidae: Saicinae) from Colombia with the description of two new species.

Tagalis Stål is recorded for the first time from Colombia and two species are described as new: Tagalis dichroa sp. nov., from Acandí (Chocó), and Tagalis albispina sp. nov., from Gorgona Island (Cauca). Based on the structure of the male genitalia of the known species, we here propose at least four species groups within Tagalis: 1) T. evavilmae, 2) T. femorata, 3) T. baenai, T. grossii, and T. marquesi, and 4) T. dichroa sp. nov., T. inornata, and T. seminigra. Tagalis albispina sp. nov. known only from the female holotype, cannot be placed into this scheme. Digital micrographs of the habitus and genitalia of the new species are provided. An updated key to the species of Tagalis is presented to facilitate the identification of the new species.

Microstructural Characterisation of Jute/Epoxy Quasi-Unidirectional Composites

The elastic properties of a composite can be predicted by micromechanical models based on the properties of the individual constituent materials of the composite and their geometrical characteristics. This paper presents a novel methodology using image analysis to determine (a) the fibre volume fraction and (b) the fibre orientation distribution factor of quasi-unidirectional jute fibre reinforced epoxy resin composites. For fibre volume fraction, digital micrographs were smoothed to reduce noise in the image, an intensity histogram informed selection of the threshold intensity for conversion to a binary image, the image was morphologically closed and opened to remove internal voids and small features respectively and the fibre volume fraction was calculated as the ratio of the detected fibre area to the total image area. For fibre orientation, the image was sharpened with Contrast-Limited Adaptive Histogram Equalisation, a threshold was set for conversion to binary and then a masking image was rotated at a number of seed points over the image to find the angles with the minimum sum of intensity at each point. The data generated was then used to validate new rules-of-mixture equations for natural fibre composites.

Automated classification of four types of developmental odontogenic cysts

Odontogenic cysts originate from remnants of the tooth forming epithelium in the jaws and gingiva. There are various kinds of such cysts with different biological behaviours that carry different patient risks and require different treatment plans. Types of odontogenic cysts can be distinguished by the properties of their epithelial layers in H&E stained samples. Herein we detail a set of image features for automatically distinguishing between four types of odontogenic cyst in digital micrographs and evaluate their effectiveness using two statistical classifiers – a support vector machine (SVM) and bagging with logistic regression as the base learner (BLR). Cyst type was correctly predicted from among four classes of odontogenic cysts between 83.8% and 92.3% of the time with an SVM and between 90±0.92% and 95.4±1.94% with a BLR. One particular cyst type was associated with the majority of misclassifications. Omission of this cyst type from the data set improved the classification rate for the remaining three cyst types to 96.2% for both SVM and BLR.

Thermal degradation, flame retardance and mechanical properties of thermoplastic polyurethane composites based on aluminum hypophosphite

Aluminum hypophosphite (AP) was used to prepare flame-retarded thermoplastic polyurethane (FR-TPU) composites, and their flame retardancy, thermal degradation and mechanical properties were investigated by limiting oxygen index (LOI), vertical burning test (UL-94), thermogravimetric analysis (TGA), cone calorimeter (CC) test, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and tensile test. TPU containing 30 wt% of AP could reach a V-0 rating in the UL-94 test, and its LOI value was 30.2. TGA tests revealed that AP enhanced the formation of residual chars at high temperatures, and slightly affected the thermal stability of TPU at high temperatures. The combustion tests indicated that AP affected the burning behavior of TPU. The peak of heat release rate (PHRR), total heat release (THR) and mass loss rate (MLR) greatly reduced due to the incorporation of AP. The tensile test results showed that both the tensile strength and the elongation at break slightly decreased with the addition of AP. The digital photos and SEM micrographs vitrified that AP facilitated the formation of more compact intumescent char layer. Based on these results mentioned above, the flame-retarding mechanism of AP was discussed. Both the self-charring during the decomposing process of AP and its facilitation to the charring of TPU led to the great improvement in the flame retardancy of TPU.

The validation of photoshop-based quantitative analysis of digital electron micrographs of developing erythrons

Introduction Erythrons are organized into erythroblastic islands. Tracing the course of cellular differentiation requires a reliable method for quantification of the morphological characteristics of these cells as they are prone to adopting highly changing phenotypes. Aim of the work The primary objective of this study was to show the applicability of Adobe Photoshop software in cytomorphometry, and the second objective was to summarize the ultrastructural characteristics of the cells forming the erythroblastic islands. Materials and methods Ultrathin sections of bone marrow were prepared from 10 albino rats and then the digitized electron microscopy images of erythrons were stored. Adobe Photoshop was used to measure the cytomorphometric parameters in the electron micrographs of these developing cells. The area occupied by any cellular component could be specified and compared directly from the histogram using the ‘Magnetic Lasso Tool’. The area pixel size and luminosity of all pixels within the selected area were recorded and the data were subjected to statistical analysis. Results The study reports a straightforward pixel-based cytomorphometric method that gives objective information about pixel values of area size and luminescence. The recorded analytical values of a nucleated erythron differed significantly from those of its preceding stage, and the average cytoplasmic luminosity of mature RBCs was 67.62% that of reticulocytes. In addition, the developmental features of erythrons were explored. Conclusion When the input material is standardized, Adobe Photoshop gives affordable and accurate measurement options that may be available for a diverse range of histomorphometric applications.

S19 Increased CRTH2 expression in asthmatic bronchial epithelium

Introduction and ObjectivesProstaglandin-D2 (PGD2) mediates chemotaxis of Th2 cells, basophils and eosinophils through the chemoattractant receptor homologous-molecule expressed on T-helper-type-2 cells (CRTh2). Pulmonary PGD2 levels are increased in patients with...

Computer-assisted counting of retinal cells by automatic segmentation after TV denoising

BackgroundQuantitative evaluation of mosaics of photoreceptors and neurons is essential in studies on development, aging and degeneration of the retina. Manual counting of samples is a time consuming procedure while attempts to automatization are subject to various restrictions from biological and preparation variability leading to both over- and underestimation of cell numbers. Here we present an adaptive algorithm to overcome many of these problems.Digital micrographs were obtained from cone photoreceptor mosaics visualized by anti-opsin immuno-cytochemistry in retinal wholemounts from a variety of mammalian species including primates. Segmentation of photoreceptors (from background, debris, blood vessels, other cell types) was performed by a procedure based on Rudin-Osher-Fatemi total variation (TV) denoising. Once 3 parameters are manually adjusted based on a sample, similarly structured images can be batch processed. The module is implemented in MATLAB and fully documented online.ResultsThe object recognition procedure was tested on samples with a typical range of signal and background variations. We obtained results with error ratios of less than 10% in 16 of 18 samples and a mean error of less than 6% compared to manual counts.ConclusionsThe presented method provides a traceable module for automated acquisition of retinal cell density data. Remaining errors, including addition of background items, splitting or merging of objects might be further reduced by introduction of additional parameters. The module may be integrated into extended environments with features such as 3D-acquisition and recognition.

Time‐dependent reduction of structural complexity of the buccal epithelial cell nuclei after treatment with silver nanoparticles

Recent studies have suggested that silver nanoparticles (AgNPs) may affect cell DNA structure in in vitro conditions. In this paper, we present the results indicating that AgNPs change nuclear complexity properties in isolated human epithelial buccal cells in a time-dependent manner. Epithelial buccal cells were plated in special tissue culture chamber / slides and were kept at 37°C in an RPMI 1640 cell culture medium supplemented with L-glutamine. The cells were treated with colloidal silver nanoparticles suspended in RPMI 1640 medium at the concentration 15 mg L⁻¹. Digital micrographs of the cell nuclei in a sample of 30 cells were created at five different time steps: before the treatment (controls), immediately after the treatment, as well as 15 , 30 and 60 min after the treatment with AgNPs. For each nuclear structure, values of fractal dimension, lacunarity, circularity, as well as parameters of grey level co-occurrence matrix (GLCM) texture, were determined. The results indicate time-dependent reduction of structural complexity in the cell nuclei after the contact with AgNPs. These findings further suggest that AgNPs, at concentrations present in today's over-the-counter drug products, might have significant effects on the cell genetic material.

Effects of Ghrelin on the Structural Complexity of Exocrine Pancreas Tissue Architecture

Recent studies have shown that ghrelin increases pancreatic exocrine secretion. However, the potential effects of ghrelin on the morphology of exocrine pancreas (EP) remain unknown. In this work, using fractal analysis, we demonstrate that centrally administered ghrelin increases structural complexity and tissue disorder in rat EP. The study was carried out on a total of 40 male Wistar rats divided into four groups (n = 10): ghrelin-treated animals (average age, 1.5 months), ghrelin-treated animals (8.5 months), and controls (1.5 and 8.5 months). The pancreas tissue sections were stained with hematoxylin/eosin and visualized by light microscopy. For each animal, the average values of tissue fractal dimension, lacunarity, as well as parameters of co-occurrence matrix texture, were determined using tissue digital micrographs. The results indicate that ghrelin administration increases EP fractal dimension and textural entropy, and decreases lacunarity, regardless of the age. To our knowledge, this is the first study to investigate the effects of ghrelin on the morphological properties of pancreatic tissue, and also the first to apply fractal and textural analysis methods in quantification of EP tissue architecture.

A novel framework for the quantitative analysis of high resolution transmission electron micrographs of soot I. Improved measurement of interlayer spacing

The reliable and reproducible quantitative image analysis of digital micrographs from high resolution transmission electron microscopy (HRTEM) of soot has been an area of interest since the early nineties. Since the resolution of HRTEM images is usually sufficient to carry out structural measurements at the atomic level, the information obtained from these images is very valuable as it potentially yields insight into very specific soot oxidation processes; however, extracting physically meaningful, reliable, accurate and statistically robust data from HRTEM images is not an easy process. Data extraction is hindered by the presence of overlapping structures, varying focus, contrast, illumination levels and noise in the images. In this paper a novel image analysis framework is presented to address these issues and explore the possibility of the extraction of high-fidelity structural data from HRTEM soot images. Emphasis is on the analysis of images of mostly amorphous, poorly ordered soot structures, as these are the most difficult to analyze.

Quantitative measurement of wound architecture in microincision cataract surgery

To evaluate whether changes in internal wound architecture after phacoemulsification can be measured quantitatively by analysis of scanning electron micrographs. Baylor College of Medicine, Houston, Texas, USA. Experimental study. Two comparative studies in human cadaver eyes were performed using coaxial small-incision cataract surgery (SICS), bimanual microincision cataract surgery (MICS), coaxial MICS, and a variety of phacoemulsification tips (MicroSurgical Technologies, Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool). After surgery, the cornea and scleral rims were harvested and digital scanning electron micrographs were taken. The internal corneal wound was analyzed using measurement software to determine the area of endothelial cell loss and length of Descemet membrane tears. Quantifiable differences were observed between combinations of techniques and tip designs. The mean area of endothelial cell loss was 2.93 ± 0.31 mm(2) (SD) after coaxial MICS (n = 4) and 2.85 ± 0.54 mm(2) after bimanual MICS (n = 4). However, after normalizing for differences in tip or sleeve diameter, the area of endothelial cell loss ranged from 1.4-fold to 1.7-fold less with coaxial SICS than with bimanual MICS with Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool tips. The mean total length of tearing was 1.38 ± 0.38 mm for bimanual MICS (n = 4) and 0.84 ± 0.61 mm for coaxial MICS (n = 4). The length and area of corneal wounds could be quantitated with accuracy and the measurements could be used to make quantifiable comparisons of phacoemulsification techniques and tips. Dr. Dimalanta is an employee of Alcon Research, Ltd. No other author has a financial or proprietary interest in any material or method mentioned.

Comparative genitalic morphology in the New World resin bugs Apiomerini (Hemiptera, Heteroptera, Reduviidae, Harpactorinae)

Apiomerini, also called New World resin bugs, are a largely Neotropical tribe of the assassin bug subfamily Harpactorinae, whose members show unique predation and maternal care behaviors. Species in this group collect resins to improve prey capture, and species within Apiomerus also coat their eggs with the accumulated resin to prevent egg predation and desiccation. Current understanding of phylogenetic relationships within the tribe is limited, in part due to the lack of comprehensive comparative morphological studies that would allow for cladistic analyses. Here, the male and female ectodermal genitalic structures are documented for all but one of the 12 extant genera of Apiomerini, including 12 species of the speciose genus Apiomerus. Descriptions and digital micrographs are provided for the pygophore, parameres, and phallus of the male, and for the tergite 8, syntergite 9/10, gonocoxae, gonapophyses, gonoplac, and bursa copulatrix of the female. Primary homologs are identified and their variation among taxa discussed. The wealth of morphological character information uncovered in this comparative analysis will greatly facilitate future cladistic analyses of Apiomerini. Our study also represents one of the first detailed and well-illustrated comparative studies of genitalic features at the tribal level in Reduviidae. Functional morphology of apiomerine genitalia is discussed where appropriate. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

A new species of Aculops (Acari: Prostigmata: Eriophyidae) from Serbia on Dipsacus laciniatus L. (Dipsacaceae), a weed target of classical biological control in the United States of America

A new eriophyoid mite species, Aculops orlovacae n. sp. (Acari: Prostigmata: Eriophyidae) collected from Dipsacus laciniatus L. (Dipsacaceae) in northern Serbia, is described and illustrated, including digital micrographs depicting key morphological characters. Differential diagnosis is provided in comparison with Aculops salixis Xue, Song et Hong, Aculops rhodensis (Keifer), Aculops hussongi Keifer and Aculops oblongus (Nalepa). This is the first eriophyoid mite species in the genus Aculops described from a host plant in the family Dipsacaceae and it is only the second eriophyoid known from a host species in the genus Dipsacus L. This mite was found during surveys for natural enemies of Dipsacus spp., as part of a classical biological control program.

Kinetics of changes in the coefficient of transmission of the adipose tissue in vitro as a result of photodynamic action

Changes in the light transmission coefficient of human adipose cells as a result of photodynamic action have been detected and investigated. Based on the statistical processing of digital micrographs of thin tissue slices, the kinetics of processes occurring in the adipose tissue has been quantitatively estimated. It has been suggested that the effects observed are brought about through the lipolysis of adipose cells with the retention of their structure.

Seasonal growth of the attachment clamps of a Paradiplozoon sp. as depicted by statistical shape analysis

Geometric morphometric methods using computer software is a more statistically powerful method of assessing changes in the anatomy than are traditional measurements of lengths. The aim of the study was to investigate whether changes in the size and shape Paradiplozoon sp. permanent attachment clamps could be used to determine the duration of the organsism’s life-cycle in situ. A total of 149 adult Paradiplozoon sp. ectoparasites were recovered from Labeobarbus aeneus and Labeobarbus kimberlyensis in the Vaal Dam. The software tool tpsDIG v.2.1 was used on six digitised landmarks placed at the junctures between the sclerites of the attachment clamps from digital micrographs. The tpsSmall v. 2.0 and Morphologika 2 v. 2.5 software tools were used to perform principal component analysis (PCA) on this multivariate dataset. The PCA analysis indicated that the increase in size and linear change in shape of the selected landmarks, were significant predictors of the sampling season. This study suggests that it takes one year for the permanent attachment clamps of a Paradiplozoon sp. to grow to their maximum size in the Vaal Dam. Key words: Diplozoidae, Labeobarbus aeneus, Labeobarbus kimberlyensis , Vaal Dam, morphometrics.

Mitotic Figure Recognition: Agreement among Pathologists and Computerized Detector

Despite the prognostic importance of mitotic count as one of the components of the Bloom – Richardson grade [3], several studies ([2, 9, 10]) have found that pathologists’ agreement on the mitotic grade is fairly modest. Collecting a set of more than 4,200 candidate mitotic figures, we evaluate pathologists' agreement on individual figures, and train a computerized system for mitosis detection, comparing its performance to the classifications of three pathologists. The system’s and the pathologists’ classifications are based on evaluation of digital micrographs of hematoxylin and eosin stained breast tissue. On figures where the majority of pathologists agree on a classification, we compare the performance of the trained system to that of the individual pathologists. We find that the level of agreement of the pathologists ranges from slight to moderate, with strong biases, and that the system performs competitively in rating the ground truth set. This study is a step towards automatic mitosis count to accelerate a pathologist's work and improve reproducibility.

Nailfold capillaroscopy by digital microscope in an Indian population with systemic sclerosis

Nailfold capillaroscopy (NFC) is a simple, non-invasive method with exceptional predictive value for the analysis of microvascular abnormalities, especially in systemic sclerosis (SSc) but remains underutilized due to cost factors of the nailfold videocapillaroscope, lack of expertise and availability issues. The aim of this study was to establish the utility of an inexpensive digital microscope to study NFC changes in SSc in correlation with disease subsets and extent of skin involvement. Twenty-two diffuse cutaneous SSc (DSS), 20 limited cutaneous SSc (LSS) patients and 42 controls were evaluated with NFC using a digital microscope at 30× and 100× magnification. Digital micrographs were used to study qualitative and quantitative changes in microvasculature. The capillary density was significantly less in all cases of SSc as compared to controls (5.3 ± 1.4 vs. 8.7 ± 1.2; P < 0.00001). Disorganized architecture was much more prevalent in DSS versus LSS (86.4%vs. 25%). The vascular deletion score (VDS) was significantly higher in DSS as compared to LSS (P < 0.0001). Scleroderma pattern (SP) was seen in 18 (81.9%) and 15 (75%) of patients with DSS and LSS, respectively. Only 4% of normal subjects showed non-specific pattern and none showed SP. The mean modified Rodnan skin score (MRSS) was positively correlated with vascular deletion score (r = 0.572; P < 0.001) and negatively with capillary density (r = -0.8; P < 0.001). Nailfold capillaroscopy changes in SSc are related to disease subset and MRSS. NFC with digital microscope is a simplified, inexpensive, outpatient procedure with results comparable to previous studies.

Mitotic figure recognition: agreement among pathologists and computerized detector.

Despite the prognostic importance of mitotic count as one of the components of the Bloom – Richardson grade [3], several studies ([2, 9, 10]) have found that pathologists’ agreement on the mitotic grade is fairly modest. Collecting a set of more than 4,200 candidate mitotic figures, we evaluate pathologists' agreement on individual figures, and train a computerized system for mitosis detection, comparing its performance to the classifications of three pathologists. The system’s and the pathologists’ classifications are based on evaluation of digital micrographs of hematoxylin and eosin stained breast tissue. On figures where the majority of pathologists agree on a classification, we compare the performance of the trained system to that of the individual pathologists. We find that the level of agreement of the pathologists ranges from slight to moderate, with strong biases, and that the system performs competitively in rating the ground truth set. This study is a step towards automatic mitosis count to accelerate a pathologist's work and improve reproducibility.

Neurongrowth, a software for automatic quantification of neurite and filopodial dynamics from time‐lapse sequences of digital images

We developed NeuronGrowth, a software for the automatic quantification of extension and retraction of neurites and filopodia, from time-lapse sequences of two-dimensional digital micrographs. NeuronGrowth requires a semiautomatic characterization of individual neurites in a reference frame, which is then used for automatic tracking and measurement of every neurite over the whole image sequence. Modules for sequence alignment, background subtraction, flat field correction, light normalization, and cropping have been integrated to improve the quality of the analysis. Moreover, NeuronGrowth incorporates a deconvolution filter that corrects the shadow-cast effect of differential interference contrast (DIC) images. NeuronGrowth was tested by analyzing the formation of outgrowth patterns by individual leech neurons cultured under two different conditions. Phase contrast images were obtained from neurons plated on CNS homogenates and DIC images were obtained from similar neurons plated on ganglion capsules as substrates. Filopodia were measured from fluorescent growth-cones of chick dorsal root ganglion cells. Quantitative data of neurite extension and retraction obtained by three different users applying NeuronGrowth and two other manually operated software packages were similar. However, NeuronGrowth required less user participation and had a better time performance when compared with the other software packages. NeuronGrowth may be used in general to quantify the dynamics of tubular structures such as blood vessels. NeuronGrowth is a free plug-in for the free software ImageJ and can be downloaded along with a user manual, a troubleshooting section and other information required for its use from http://www.ifc.unam.mx or http://www.ifc.unam.mx/ffm/index.html.