Quantification Of Morphological Changes Research Articles

Metal-Nano-Ink Coating for Monitoring and Quantification of Cotyledon Epidermal Cell Morphogenesis.

During cotyledon growth, the pavement cells, which make up most of the epidermal layer, undergo dynamic morphological changes from simple to jigsaw puzzle-like shapes in most dicotyledonous plants. Morphological analysis of cell shapes generally involves the segmentation of cells from input images followed by the extraction of shape descriptors that can be used to assess cell shape. Traditionally, replica and fluorescent labeling methods have been used for time-lapse observation of cotyledon epidermal cell morphogenesis, but these methods require expensive microscopes and can be technically demanding. Here, we propose a silver-nano-ink coating method for time-lapse imaging and quantification of morphological changes in the epidermal cells of growing Arabidopsis thaliana cotyledons. To obtain high-resolution and wide-area cotyledon surface images, we placed the seedlings on a biaxial goniometer and adjusted the cotyledons, which were coated by dropping silver ink onto them, to be as horizontal to the focal plane as possible. The omnifocal images that had the most epidermal cell shapes in the observation area were taken at multiple points to cover the whole surface area of the cotyledon. The multi-point omnifocal images were automatically stitched, and the epidermal cells were automatically and accurately segmented by machine learning. Quantification of cell morphological features based on the segmented images demonstrated that the proposed method could quantitatively evaluate jigsaw puzzle-shaped cell growth and morphogenesis. The method was successfully applied to phenotyping of the bpp125 triple mutant, which has defective pavement cell morphogenesis. The proposed method will be useful for time-lapse non-destructive phenotyping of plant surface structures and is easier to use than the conversional methods that require fluorescent dye labeling or transformation with marker gene constructs and expensive microscopes such as the confocal laser microscope.

Comprehensive assessment of myocardial remodeling in ischemic heart disease by synchrotron propagation based X-ray phase contrast imaging

Cardiovascular research is in an ongoing quest for a superior imaging method to integrate gross-anatomical information with microanatomy, combined with quantifiable parameters of cardiac structure. In recent years, synchrotron radiation-based X-ray Phase Contrast Imaging (X-PCI) has been extensively used to characterize soft tissue in detail. The objective was to use X-PCI to comprehensively quantify ischemic remodeling of different myocardial structures, from cell to organ level, in a rat model of myocardial infarction. Myocardial infarction-induced remodeling was recreated in a well-established rodent model. Ex vivo rodent hearts were imaged by propagation based X-PCI using two configurations resulting in 5.8 µm and 0.65 µm effective pixel size images. The acquired datasets were used for a comprehensive assessment of macrostructural changes including the whole heart and vascular tree morphology, and quantification of left ventricular myocardial thickness, mass, volume, and organization. On the meso-scale, tissue characteristics were explored and compared with histopathological methods, while microstructural changes were quantified by segmentation of cardiomyocytes and calculation of cross-sectional areas. Propagation based X-PCI provides detailed visualization and quantification of morphological changes on whole organ, tissue, vascular as well as individual cellular level of the ex vivo heart, with a single, non-destructive 3D imaging modality.

Microglial dynamics during brain development.

Microglial dynamics during brain development.

Deep learning approaches in electron microscopy imaging for mitochondria segmentation

Deep neural networks provide outstanding classification and detection accuracy in biomedical imaging applications. We present a study for mitochondria segmentation in electron microscopy (EM) images. Mitochondria play a significant role in cell cycle by generating the needed energy, and show quantifiable morphological differences with diseases such as cancer, metabolic disorders, and neurodegeneration. EM imaging allows researchers to observe the morphological changes in cells as part of disease process at a high resolution. Manual segmentation of mitochondria in large sequences of EM images is time consuming and prone to subjective delineation. Thus, manual segmentation may not provide the high accuracy needed for accurate quantification of morphological changes. We show that a convolutional neural network provides accurate mitochondria segmentation in CA1 hippocampus area of brain that is imaged by a focused ion beam scanning electron microscope (FIBSEM). We compare our results with other studies which report results on the same data set and with other deep neural network approaches, and provide quantitative comparison.

Enhanced activation of eosinophils in peripheral blood and implications for eosinophilic esophagitis diagnosis

Eosinophils are markers of the eosinophilic esophagitis (EoE) disease, and this work aimed to assess whether activation of eosinophils could be a noninvasive test to contribute for EoE diagnosis. The activation state of peripheral blood eosinophils in EoE patients and control subjects was assessed based on the morphological aspects of the eosinophil after adherence to slide. Cyclooxygenase-2 and 5-lipoxygenase expressions were evaluated by means of immunofluorescence microscopy to verify if and which eicosanoid pathway is triggered in eosinophils in blood in EoE. The eosinophils of patients with EoE were significantly more activated than those of control individuals. The lowest percentage of normal eosinophils for control subjects was 40%, while the highest percentage of eosinophils of normal aspect for patients with EoE was 32%. Considering 36% as a cutoff for normal eosinophils, this value differentiated all individuals with EoE from individuals without the disease with a sensitivity of 100%, considering the diagnosis of EoE as currently defined. Eosinophils of EoE patients showed higher expression of cyclooxygenase-2 than those of control subjects. The quantification of morphological changes in eosinophils is a feasible, easy, and reliable manner to identify EoE patients. Therefore, patients with symptoms of esophageal dysfunction showing higher than 36% activated eosinophils in peripheral blood could be a useful way to help definition and diagnostic criterion for EoE.

Quantification of Pulmonary Fibrosis in a Bleomycin Mouse Model Using Automated Histological Image Analysis.

Current literature on pulmonary fibrosis induced in animal models highlights the need of an accurate, reliable and reproducible histological quantitative analysis. One of the major limits of histological scoring concerns the fact that it is observer-dependent and consequently subject to variability, which may preclude comparative studies between different laboratories. To achieve a reliable and observer-independent quantification of lung fibrosis we developed an automated software histological image analysis performed from digital image of entire lung sections. This automated analysis was compared to standard evaluation methods with regard to its validation as an end-point measure of fibrosis. Lung fibrosis was induced in mice by intratracheal administration of bleomycin (BLM) at 0.25, 0.5, 0.75 and 1 mg/kg. A detailed characterization of BLM-induced fibrosis was performed 14 days after BLM administration using lung function testing, micro-computed tomography and Ashcroft scoring analysis. Quantification of fibrosis by automated analysis was assessed based on pulmonary tissue density measured from thousands of micro-tiles processed from digital images of entire lung sections. Prior to analysis, large bronchi and vessels were manually excluded from the original images. Measurement of fibrosis has been expressed by two indexes: the mean pulmonary tissue density and the high pulmonary tissue density frequency. We showed that tissue density indexes gave access to a very accurate and reliable quantification of morphological changes induced by BLM even for the lowest concentration used (0.25 mg/kg). A reconstructed 2D-image of the entire lung section at high resolution (3.6 μm/pixel) has been performed from tissue density values allowing the visualization of their distribution throughout fibrotic and non-fibrotic regions. A significant correlation (p<0.0001) was found between automated analysis and the above standard evaluation methods. This correlation establishes automated analysis as a novel end-point measure of BLM-induced lung fibrosis in mice, which will be very valuable for future preclinical drug explorations.

Immunomorphological characteristics of the synovial membrane in rheumatic diseases

The synovial membrane is frequently a target in rheumatic diseases. A search for diagnostic criteria and determination of changes in the pathological process necessitate standardized biopsy diagnostic techniques and quantification of morphological changes using digital imaging methods. The paper considers main methods for obtaining synovial membrane samples. It presents major morphological and immunohistochemical variations in synovitis in the presence of rheumatoid arthritis, ankylosing spondylitis, and osteoarthrosis. It shows different immunological and autoinflammatory mechanisms of these diseases. Synovial membrane inflammation in rheumatoid arthritis, ankylosing spondylitis, and osteoarthrosis is characterized by different components of morphogenesis, which is proven by the expression of different cell markers. Rheumatoid synovitis is an autoinflammatory process; synovitis in ankylosing spondylitis is characterized by autoinflammatory processes; biomechanical factors as joint inflammation triggers are leading in osteoarthrosis.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Aligning 3D time-of-flight MRA datasets for quantitative longitudinal studies: evaluation of rigid registration techniques

Objective3D Time-of-flight (TOF) magnetic resonance angiography is commonly used for vascular analyses. A quantification of longitudinal morphological changes usually requires the registration of TOF image sequences acquired at different time points. The aim of this study was to evaluate the precision of different 3D rigid registration setups such that an optimal quantification of morphological changes can be achieved. MethodsEight different rigid registration techniques were implemented and evaluated in this study using the target registration error (TRE) calculated based on 554 landmarks defined in twenty TOF datasets. The registration techniques differed in integration of brain and vessel segmentation masks and usage of a multi-resolution framework. Furthermore, the benefit of a prior volume-of-interest definition for registration accuracy was evaluated. ResultsThe results revealed that the highest registration accuracies can be achieved using a multi-resolution framework and a cerebrovascular segmentation as mask. Numerically, a mean TRE of 1.1mm was calculated. If applicable, a prior definition of a volume-of-interest allows a reduction of the TRE to only 0.6mm. ConclusionTOF datasets should be registered using vessel segmentations as mask, multi-resolution framework and previous volume-of-interest definition if possible to obtain the highest registration precision. This is especially the case for longitudinal datasets that are separated by several months while the registration technique seems less important for datasets that are only separated by a few days.

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology even in complex tissue sections. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgenössische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

Quantitative and qualitative analysis of heart mitochondria for evaluating the degree of myocardial injury utilizing atomic force microscopy

Mitochondrial dysfunction plays a central role in mediating both the necrotic and apoptotic components of reperfusion injury. Because mitochondrial swelling is one of the most important indicators of the beginning of mitochondrial permeability transition, quantification of morphological changes in mitochondria would be useful in evaluating the degree of IR injury, as well as the protective effects of various therapies. In this study, we characterized the morphological changes in heart mitochondria caused by the duration and severity of ischemia utilizing particle shape analysis on atomic force microscopy (AFM) topographic images. We also simultaneously investigated the nano-mechanical changes in rat heart mitochondria by injury using force–distance curve measurements. Rats were randomly divided into 3 groups: control group (n=3), myocardial ischemia without reperfusion (PI group, n=3), and myocardial ischemia with reperfusion (IR group, n=4). Normal mitochondria appeared ellipsoidal with a mean area of 3551±1559nm2 and mean perimeter of 217.54±52.09nm (n=60). The mean area and perimeter of mitochondria in the IR groups increased to 28,181±21,248nm2 and 595.74±234.29nm (n=40, p<0.0001 vs. control group, respectively), maintaining oval in shape. But, in the PI group, all parameters showed significant differences compared to parameters of the control group (n=35, p<0.0001). In particular, the mean axial ratio and roundness were significantly different from those in the IR group. Mitochondria in the PI group looked more spherical than those of control and IR groups. Adhesion force is the force before the last event on the retraction half of force–distance curve measurements, corresponding to the point where the tip and the surface loose contact. The adhesion forces of heart mitochondria in the IR and PI groups significantly decreased to 19.56±1.08nN (n=30, p<0.0001) and 18.65±3.18nN (n=30, p<0.0001), compared to normal mitochondria which had an adhesion force of 27.64±0.88nN (n=30). Adhesion force is governed by the attractive portion of the interacting forces between the surface atoms of the contacts. From the morphological and nano-mechanical changes in heart mitochondria, we suggested that the outer membranes of mitochondria were broken by myocardial ischemic injury before they became swollen, and the swelling might be correlated with the ischemic injury. We inferred that the breakage of membranes leads to uptake of water and matrix swelling. As a result, shape measurement parameters for the quantitative analysis of mitochondrial swelling could be very effective for evaluating the myocardial injury.

Quantification of Changes in Morphology, Mechanotransduction, and Gene Expression in Bovine Articular Chondrocytes in Response to 2-Dimensional Culture Indicates the Existence of a Novel Phenotype.

Objective:Matrix-induced autologous chondrocyte implantation (ACI) offers a potential solution for cartilage repair but is currently hindered by loss of the chondrocyte differentiated phenotype. To further our understanding of the mechanism of dedifferentiation, changes in the phenotype in relation to mechanotransduction were recorded in response to monolayer culture.Methods:Bovine cartilage explants were excised and chondrocytes cultured for 9 days (P1), 14 days (P2), and 21 (P3) days. Changes in morphology and regulatory volume increase (RVI; a mechanotransduction response) were determined by the expression of key genes by RT-PCR and confocal microscopy, respectively.Results:A loss of a differentiated phenotype was observed in P1 with a reduction in sphericity and an overall increase in cell volume from 474.7 ± 32.1 µm3 to 725.2 ± 35.6 µm3. Furthermore, the effect of 2-dimensional (2-D) culture-induced dedifferentiation on mechanotransduction was investigated, whereby RVI and Gd3+-sensitive REV5901-induced calcium rise were only observed in 2-D cultured chondrocytes. A significant up-regulation of types I and II collagens and Sox9 was observed in P1 chondrocytes and no further significant change in type I collagen but a return to baseline levels of type II collagen and Sox9 upon further culture.Conclusion:These data indicated the presence of an intermediate, mesodifferentiated phenotype and highlight the importance of mechanotransduction as a marker of the chondrocytic cell type.

Altered Islet Composition and Disproportionate Loss of Large Islets in Patients with Type 2 Diabetes

Human islets exhibit distinct islet architecture with intermingled alpha- and beta-cells particularly in large islets. In this study, we quantitatively examined pathological changes of the pancreas in patients with type 2 diabetes (T2D). Specifically, we tested a hypothesis that changes in endocrine cell mass and composition are islet-size dependent. A large-scale analysis of cadaveric pancreatic sections from T2D patients (n = 12) and non-diabetic subjects (n = 14) was carried out combined with semi-automated analysis to quantify changes in islet architecture. The method provided the representative islet distribution in the whole pancreas section that allowed us to examine details of endocrine cell composition in individual islets. We observed a preferential loss of large islets (>60 µm in diameter) in T2D patients compared to non-diabetic subjects. Analysis of islet cell composition revealed that the beta-cell fraction in large islets was decreased in T2D patients. This change was accompanied by a reciprocal increase in alpha-cell fraction, however total alpha-cell area was decreased along with beta-cells in T2D. Delta-cell fraction and area remained unchanged. The computer-assisted quantification of morphological changes in islet structure minimizes sampling bias. Significant beta-cell loss was observed in large islets in T2D, in which alpha-cell ratio reciprocally increased. However, there was no alpha-cell expansion and the total alpha-cell area was also decreased. Changes in islet architecture were marked in large islets. Our method is widely applicable to various specimens using standard immunohistochemical analysis that may be particularly useful to study large animals including humans where large organ size precludes manual quantitation of organ morphology.

Histopathology of insulin‐induced laminitis in ponies

Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin-induced laminitis. Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid-dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.

Lip impressions: a new method for monitoring morphological changes in orofacial granulomatosis

To develop and evaluate an objective method for assessing lip size and treatment-related morphological changes in orofacial granulomatosis (OFG) patients. Patients with swollen lips because of OFG (n=21) were enrolled. A light-body polyvinylsiloxane material was used to take lip impressions before and after treatment (n=10), or during treatment (n=11). Plaster models were cast from the impressions and the lips were measured using callipers. The intra-examiner and inter-examiner reproducibility of the technique were assessed. OFG patients had significantly larger lips than controls (P<0.0001). The coefficient of variation on repeated measurements of the same impression was 1.6% and for duplicate impressions was 2.6%. Significant reduction in lip size was shown in all 10 patients after diet restriction (P<0.002). Seven of 11 patients whose impressions were taken at least 3 months after the initiation of cinnamon- and benzoate-free diet also showed reduction in lip size during follow up (P<0.002). Serial lip impressions appear to be reliable for routine quantification of morphological changes of the lips in OFG patients. We present a new reproducible and sensitive method for assessing changes in lip size in response to treatment in OFG.

Morphology Evolution of an Ebb-Tidal Delta Following a Storm Perturbation: Assessments from Remote Sensed Video Data and Direct Surveys

Abstract Tidal inlet morphology is strongly dependent on local hydrodynamic conditions that rely on wave/tide dominance. In some places, this hydrodynamic context exhibits a very strong seasonal pattern, showing tide-induced morphologies in summer and wave-induced ones in winter. The Barra Nova tidal inlet in Algarve, South Portugal, presents such characteristics. Short term morphological changes were investigated during an extensive field campaign carried on in February and March 1999, after the winter storm period. This study integrates both topo-bathymetric surveys and remotely sensed video data in order to assess the morphological evolution of the inlet system during the transitional period following storms. Comparison of datasets issued from these two direct and indirect methods has shown a very good agreement (errors usually less than 10 cm in elevation) that allows the quantification of morphological changes of the entire inlet system during the whole period of video observation. During a transitio...

Scale-independent shape analysis for quantitative cytology using mathematical morphology

A system for automatic quantification of morphological changes of cell lines, proposed for cytotoxicity tests of biomaterials, is presented. Light-micrographs of cultured cells are segmented by adaptive thresholding within a local adaptive window. Connected cells in binarized micrographs are separated by a novel morphological multiscale method, treating cells in their size-specific scale and hence resulting in scale-independent separations. Significant shape descriptors correlating well with cell toxicity are extracted from single cells. Size and compactness distributions turned out to be reliable and useful parameters, providing an alternative to the common subjective grading of shape deformations by visual inspection. The system is evaluated for several standardized toxical reference substances and is now in use for clinical biocompatibility testing.

Light microscopic quantification of morphological changes during aging in neurons and glia of the rat parietal cortex.

Different changes in neuronal and glial population of the aging brain have been described; however, the degree and extent of these changes are controversial. This study evaluates the quantitative and cytomorphometric effects of aging on neuronal and glial populations in the parietal cortex of the rat. The study was performed in two groups of rats aged 4-6 and 30-32 months. Cortical volume, neuronal density, glial density, and neuronal area, and shapes of the soma and nucleus were analyzed in cortical layers I, II-IV, V, and VI using serial sections stained with cresyl-fast-violet, and quantitative morphometric techniques. No changes with age were found in volume of the cortex or neuronal density. Glial density increased significantly (mean for all layers 17%) in older rats. Layers II-IV, V, and VI showed an age-related decrease in the area of the neuronal soma. Neuronal shape, as revealed by the major/minor diameter ratio, also showed a decrease in old rats but only in layer II-IV. Nuclear area decreased with age only in layer VI. The stability of neuronal density together with the increased number of glial cells and the changes in neuronal soma size suggest that aged-related cognitive impairment could be a consequence of neuronal dysfunction rather than actual neuronal losses.

Light microscopic quantification of morphological changes during aging in neurons and glia of the rat parietal cortex

Light microscopic quantification of morphological changes during aging in neurons and glia of the rat parietal cortex