12/24/2023 0 Comments Imagej citationThese steps are achieved by drawing the ROI, using one of the drawing tools (e.g., freehand or ellipse tool) (Fig. A useful feature of ImageJ is the ROI Manager that allows selection of specific areas for evaluation, or deselecting areas of unwanted elements (Fig. ROIĪ specific ROI can be analyzed rather than the whole image. This is particularly recommended when intensity is measured for an entire image or when differing portions of separate images are to be compared. One method to correct for uneven illumination in fluorescence images has been previously described (Model and Burkhardt, 2001). When staining intensity is being quantified, a uniform background is required in all areas of the image. Thresholding tool settings that successfully quantify staining in a positive control specimen should then be duplicated in every image to be compared. Identical reagents and processing should be used, with identical image acquisition settings and exposure times. To compare multiple specimens, staining and image acquisition should be performed in parallel for the entire set. Snapshot of ImageJ user guide showing the threshold feature (p 79). Intensity measurements can also be performed within regions of interest (ROIs see below). “Analyze–Analyze Particles” can be used to measure individual feature profiles. (5) Select “Analyze–Measure.” The results table will appear, which can then be saved. The “Limit to Threshold” option should also be selected, because otherwise the entire image will be measured, rather than the selected area. Ensure that all the gray level measurements are selected. (4) Select “Analyze–Set Measurements” and choose the parameters to be measured. (3) Click on “Set” to set the threshold of the image. (2) Choose “Dark background” for fluorescence. A histogram is displayed to provide assistance. (1) Select “Image–Adjust–Threshold.” The auto setting can be selected or the sliders can bemanually moved until all the stained areas areselected. The image needs to be opened and then the following steps applied. The following information is based on a guide provided by the University of Auckland ( ). Staining can be assessed by setting a “threshold” using the thresholding tool (Fig. ImageJ will then automatically generate the immunohistochemistry index. If the researcher wishes to count stained or fluoresced cell profiles, these can be counted directly from the screen by placing marks of different colors onto positive and negative nuclei by clicking the mouse. Other formats can be opened with the aid of plugins. ImageJ opens the following file formats: TIFF (default format), JPEG, PNG, GIF, BMP, DICOM, PGM, and FITS. USING IMAGEJ TO ANALYZE IMMUNOHISTOCHEMISTRY Another option is MBF ImageJ bundle or ImageJ for Microscopy (formerly WCIF-ImageJ), featuring a collection of plugins and macros, collected and organized by Tony Collins at the MacBiophotonics Facility, McMaster University. Biologists who are unsure of which distribution to choose should opt for Fiji. Various projects address the difficult task of using ImageJ beyond the basics. ImageJ add-ons are available from multiple sources (see p 2 of the ImageJ user guide for suggestions). Hundreds of plugins are available for free downloading to ImageJ (Collins, 2007). ImageJ's strength lies in the extensive number of plugins that extend its functionality. Thereafter, captured images are opened in NIH Image/ImageJ for evaluating indices of positivity on immunohistochemistry slides, as well as fluorescence images. For the evaluation of immunohistochemistry slides using ImageJ, images are captured onto the hard drive of the workstation computer. Downloadable copies are available for Windows, Linux, and Mac OSX. Java runtime environments are available for free from Sun Microsystems or bundled with platform-specific installations of ImageJ ( /ij). ImageJ runs on any computer that is a Java 1.5 or later virtual machine. ImageJ is available in the public domain (i.e., free). ImageJ is a Java image processing and analysis program based on NIH Image for the Macintosh. The cost effective answer for quantitative immunohistochemical analysis is ImageJ, developed by WayneRasband ( ). Robert Barrett, PhD, from the University of Auckland. In this example of a fluorescent image, the researcher may statistically determine the amount of fluorescence and compare the results with a control group. The image shows gyral white matter of a 0.7 gestation fetal sheep 3 days after a 25-min umbilical cord occlusion. The 40× image was taken on a Nikon i90 microscope with a motorized stage. A 6-µm paraffin embedded section stained with IB4 and Alexa Fluor 468-conjugated antibody (green).
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