ImageJ analysis
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For decades, biologists have relied on software to visualize and interpret imaging data. As techniques for acquiring images increase in complexity, resulting in larger multidimensional datasets, imaging software must adapt. ImageJ is an open-source image analysis software platform that has aided researchers with a variety of image analysis applications, driven mainly by engaged and collaborative user and developer communities. The close collaboration between programmers and users has resulted in adaptations to accommodate new challenges in image analysis that address the needs of ImageJ's diverse user base. ImageJ consists of many components, some relevant primarily for developers and a vast collection of user-centric plugins. It is available in many forms, including the widely used Fiji distribution. We refer to this entire ImageJ codebase and community as the ImageJ ecosystem. Here we review the core features of this ecosystem and highlight how ImageJ has responded to imaging technology advancements with new plugins and tools in recent years. These plugins and tools have been developed to address user needs in several areas such as visualization, segmentation, and tracking of biological entities in large, complex datasets. Moreover, new capabilities for deep learning are being added to ImageJ, reflecting a shift in the bioimage analysis community towards exploiting artificial intelligence. These new tools have been facilitated by profound architectural changes to the ImageJ core brought about by the ImageJ2 project. Therefore, we also discuss the contributions of ImageJ2 to enhancing multidimensional image processing and interoperability in the ImageJ ecosystem.
UNLABELLED: ThunderSTORM is an open-source, interactive and modular plug-in for ImageJ designed for automated processing, analysis and visualization of data acquired by single-molecule localization microscopy methods such as photo-activated localization microscopy and stochastic optical reconstruction microscopy. ThunderSTORM offers an extensive collection of processing and post-processing methods so that users can easily adapt the process of analysis to their data. ThunderSTORM also offers a set of tools for creation of simulated data and quantitative performance evaluation of localization algorithms using Monte Carlo simulations. AVAILABILITY AND IMPLEMENTATION: ThunderSTORM and the online documentation are both freely accessible at https://code.google.com/p/thunder-storm/.
Software based analyses of immunohistochemical staining are designed for obtaining quantitative, reproducible, and objective data. However, often times only a certain type of positive cells or structures need to be quantified thus whole image analysis cannot be performed. Such an example is Hofbauer placental cells, which show positivity of some antigens together with trophoblast, but only Hofbauer cells represent the regions of interest (ROIs). Two independent observers evaluated the immunohistochemical staining intensity of Hofbauer cells in placenta samples stained for cytoplasmic antigens by ImageJ, QuPath and light microscopy. Thus, the precise manual determination of ROIs, i.e. Hofbauer cells, was necessary. We detected low inter-observer variability in staining intensity. Almost perfect agreement between observers was reached for ImageJ and QuPath whilst substantial agreement was reached for light microscopy evaluation. As for the comparison of ImageJ, QuPath and light microscopy, the agreement of all three methods (identical immunohistochemical intensity) was achieved for 38.1% samples. The almost perfect agreement of staining intensities was reached between ImageJ and QuPath, and moderate agreement for comparison of the light microscopy to both software. Software analyses are much more time-consuming, thus their utilization is at least questionable to evaluate ROIs with selection.
- Publikační typ
- časopisecké články MeSH
(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.
Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.
This chapter gives examples of basic procedures of quantification of plant structures with the use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.
Hofbauer cells are macrophages residing in the stroma of placental villi and play a number of roles during normal pregnancy, as well as pathological conditions. A morphometric analysis of Hofbauer cells, in particular to investigate the number of cells, their size and shape in samples of normal human placenta from 1st trimester, term and with chorioamnionitis was performed. Tissue samples were immunostained for CD206 antigen and evaluated using ImageJ software. We detected significant changes in number and morphology of HBCs between normal placenta and placenta with chorioamnionitis samples. In chorioamnionitis, the cells were unevenly distributed within the villi, generally present in higher numbers, larger and more elongated than those in normal 1st trimester and term placenta.
- MeSH
- chorioamnionitida * veterinární MeSH
- choriové klky MeSH
- lidé MeSH
- makrofágy MeSH
- placenta MeSH
- těhotenství MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This chapter gives examples of basic procedures of quantification of plant structures with use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are: (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.
- MeSH
- epidermis rostlin anatomie a histologie ultrastruktura MeSH
- listy rostlin anatomie a histologie ultrastruktura MeSH
- mikroskopie metody MeSH
- optické zobrazování metody MeSH
- počítačové zpracování obrazu metody MeSH
- průduchy rostlin anatomie a histologie ultrastruktura MeSH
- rostliny anatomie a histologie ultrastruktura MeSH
- software MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The cortical microtubule and actin meshworks play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM), spinning disk confocal microscopy (SDCM), and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of free computing tools based on the publicly available ImageJ software package, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.
- MeSH
- Arabidopsis ultrastruktura MeSH
- cytoskelet ultrastruktura MeSH
- fluorescenční mikroskopie metody MeSH
- konfokální mikroskopie metody MeSH
- mikrotubuly ultrastruktura MeSH
- počítačové zpracování obrazu metody MeSH
- rostlinné buňky ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The acromion projects laterally in a curving orientation. When a fracture of the acromion occurs, despite being rare, matching the best-fit fixation plate is challenging due to few options and limited availability of fixation plate types. Alternative fixation methods can carry risk of nonunion complications. PURPOSE: The objective of this study was to investigate the morphological curvature pattern of the acromion to assess the level of the fixation plates fitness and provide the suitable imaging modalities for evaluating the acromion curvature. BASIC PROCEDURES: The correlation between the acromion curvature and five fixation plates were calculated and their fitness level was evaluated statistically. The curvature of acromion and the five available fixation plates were photographed and assessed digitally by software (FIJI ImageJ and Microsoft Excel). The method entails plotting seven points along the curving surface and margins of the acromion, while the Excel Solver function calculates the regression, ultimately giving curvature values. First, the acromion parameters were studied on 180 paired healthy dry scapulae. Then, the acromion curvature values were compared to the fixation plates curvatures. Likewise, the acromion curvature was assessed as applicable on 153 (100 AP- views, 50 Y- views, and 3 superior-views) retrospective plain X-rays and 40 3D-CT scapula reconstructions of healthy acromia. MAIN FINDINGS: The mean length of the acromion was 48.70 ± 5.29 mm, mean thickness was 8.51 ± 1.67 mm, and mean width was 25.97 ± 5.97 mm. The calculated values of the mean curvatures were 0.050 ± 0.015 mm-1 for the mean acromion surface, 0.042 ± 0.027 mm-1 for the internal margin, and 0.055 ± 0.010 mm-1 for the external margin. The curved geometry of the acromion was plotted on a graph giving a spectrum of curvature patterns with distribution values revealing fixation plates fitness represented by area under the curve with frequencies of 4.32 % for the acromion-specific fixation plate, 14.28 % for the large clavicle fixation plate, 0.26 % for the small clavicle fixation plate, 53.38 % for the flexible universal fixation plate, and none for the rigid universal fixation plate. PRINCIPAL CONCLUSIONS: Approximately 27.76 % of the acromion surface curvatures distribution does not overlap with any of our measured fixation plates. Evaluating the acromion surface curvature was possible on plain X-rays in the Y-view only.
- MeSH
- akromion * diagnostické zobrazování zranění anatomie a histologie chirurgie MeSH
- fraktury kostí * chirurgie diagnostické zobrazování MeSH
- kostní destičky * MeSH
- lidé MeSH
- lopatka * diagnostické zobrazování anatomie a histologie MeSH
- radiografie MeSH
- vnitřní fixace fraktury * metody přístrojové vybavení MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
