As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. You should modify the size parameter in the line below based // on the calibration of your images run( "Analyze Particles.", "size=1-Infinity circularity=0-1.When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration. Run( "Set Measurements.", "area mean perimeter shape limit redirect=None decimal=4") Get the filename from the title of the image that's open for adding to the results table // We do this instead of using the imageFile parameter so that the // directory path is not included on the table filename = getTitle() Store the number of results before executing the commands, // so we can add the filename just to the new results prevNumResults = nResults Save the results data saveAs( "results", outputFile)
Multi measure plugin imagej update#
SetBatchMode( false) // Now disable BatchMode since we are finished updateResults() // Update the results table so it shows the filenames // Show a dialog to allow user to save the results file outputFile = File. Get the list of files from that directory // NOTE: if there are non-image files in this directory, it may cause the macro to crash fileList = getFileList( inputDirectory) Show the user a dialog to select a directory of images inputDirectory = getDirectory( "Choose a Directory of Images") Macro to measure Area, Intensity, Perimeter, and Shape of directory of images run( "Clear Results") // clear the results table of any previous measurements // The next line prevents ImageJ from showing the processing steps during // processing of a large number of images, speeding up the macro setBatchMode( true) We will call the function “processImage” and it will contain the lines that were recorded using the macro recorder.
Multi measure plugin imagej code#
The Macro code box will pop up.įirst, we will create a function to execute the steps we just recorded. To create a new macro, on the ImageJ menu, click Plugins > New > Macro. The second line actually calls the Analyze Particles command and gives it the parameters needed to execute the function, this is the line we need. The first line where this appears is not necessary, since that line will show the dialog box for the Analyze Particles window, so you can delete that line, since for automating the analysis of a whole plate, we don’t want to show the Analyze Particles dialog for every image. Also notice that there are 2 lines which show the run(“Analyze Particles…”) command. The first line is commented out (everything on a line “//” in ImageJ code is ignored, known as a comment), since that line would show the Threshold dialog box, and we want to set the threshold automatically, not show the dialog box for user input. The results table will pop up, showing you 4 rows, one row for each cell detected. Select “Display results” since we want to save the results to the results table after analysis.
We enter 1 um² as the lower limit, that way we will eliminate any specks of dust that show up in the image. For the size parameter, enter in 1.00-Infinity. The Analyze Particles dialog will pop up. Click the Analyze menu, and then select Analyze Particles. The next step is to use the Analyze Particles function in ImageJ to detect the cells and measure their characteristics. Also select Limit to threshold, otherwise ImageJ will calculate this measurements on the whole image instead of the thresholded areas, which is not what we want to do. Select Area, Mean gray value, Perimeter, and Shape descriptors (for circularity measurement). On the ImageJ menu, select Analyze > Set Measurements. Next we need to select the measurements we want to obtain from the particles.
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