![]() Run("Set Measurements.", "area mean min centroid fit redirect=None decimal=1") Run("Duplicate.", "title=HumanXLCytokineArray_forThres.tif") SelectWindow("TransformedHumanXLCytokineArray.tif") threshold the transformed mask (rotation creates non-binary image) Run("Landmark Correspondences", "source_image=HumanXLCytokineArray.tif template_image= transformation_method= alpha=1 mesh_resolution=32 transformation_class=Affine interpolate") run Landmark Correspondence to obtain a rough mask WaitForUser("Select 4-5 landmarks in each image:\n1) Select the corresponding landmarks in the same order in the blot to be analyzed and the template.\n2) Click OK ") get starting mask using "Landmark Correspondences" register the mask to the image of the dot-blot ") ĭialog.addNumber("Spot diameter (px)", 14) spotsize: all spot-ROIs will set to equal sizeĭialog.addMessage("The macro will create circular ROIs with equal diameter above each spot. GetDimensions(width, height, channels, slices, frames) Run("Set Scale.", "distance=0 known=0 pixel=1 unit=pixel") get dot-blot filename and path for saving SelectWindow("HumanXLCytokineArray.tif") make sure that the dot-blot is the active window, by putting behind the HumanXLCytokineArray.tif If (nImages>2) waitForUser("Please close all other images and press OK") If (nImages!=2) exit("Please open HumanXLCytokineArray.tif and your dot-blot image.") current address: BioImage Informatics Facility, SciLifeLab, Upppsala, Sweden. created by A.Klemm, at BMC, LMU Munich, Germany output: saves in the original folder: results (_results.xls), control image (_detectedWells.tif), saved roi-manager (_ROIs.zip) input: original image of membrane to be analyzed and HumanXLCytokineArray.tif subtracts background and inverts the original image before analysis. simplifies use of "Landmark Correspondences" plugin to analyse dot blots. Template (via link, problems uploading it to the forum) : You can re-open the ROI-manager by drag&drop the zip-file as it is to Fiji. The macro saves the measurement, a control-image and the ROI-manager in the same folder as your images. For this, select a ROI in the ROI-manager, move the ROI to the desired position and click “Update” in the ROI-manager.) once the ROIs are obtained you have the possible to adjust them manually, if really needed. ![]() Select the same spots in the same order in the template image. Try to select clear spots at each corner of the blot array and maybe one in the center. Select 4-5 (clearly) visible spots in your blot. Running the script you will be prompted to: Intensity measurements are done on the inverted (background black, spots bright), background-subtracted image. With the help of the template (HumanXLCytokineArray.tif), it creates circular ROIs with a given radius around each spot position. Hi you have the exact same template you can use my script below. ![]()
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