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Multicolor stack in imagej fiji3/20/2024 ![]() ![]() But you can use the Watershed tool to detect and separate these objects so they can be counted as individual objects again. This can be a problem for some types of analysis, such as counting. When you create a mask/binary of these they will appear as a single object. Sometimes separate objects in your image will be touching. Erode will remove a thin layer (1 pixel) from around the edge of the mask/binary. Dilate will add a thin layer (1 pixel) around the edge of the existing mask/binary. To add or remove from the edges of a mask or binary you can use the Dilate or Erode tools. This could be useful for things like membrane measurements or morphology analysis. Sometimes you will need to add or remove a small layer from the edges of the mask/binary. Small gaps left in the mask or binary after thresholding can easily be corrected for by using the Close tool. All of these tools can be found under Process -> Binary. There are a number of tools available to help you get the best fit to your data. Sometimes adjustments need to be made to binaries/masks before analysis to ensure it is accurately representing the data. Macs will often produce reversed background and foreground colours also. However, depending on your settings (both the general settings in FIJI, and those selected from the binary options above), the background and foreground colours can somtimes be reversed in the mask and binary. Generally masks and binaries will appear as shown in the images below. To generate a binary go to Process -> Binary -> Make Binary.įor this option, a window will open with binary options, select your options and click OK to generate the binary image. You can also go to Process -> Binary -> Convert to Mask. As shown above, if you select Apply in the threshold window it will convert the threshold to a mask. If you need to convert your threshold to a mask, or binary, there are several options you can use. The list in the log that was created will show the order of the algorithms used in the montage.įrom this montage you can decide which algorthim may suit your data the best and apply it using the steps above. Press OK.Ī montage will be generated showing what each auto threshold algorithm would produce on the image. In the dialog box that opens up set the Method to Try All, make sure the White objects on black background (for fluorescence images) and Show threshold values in log window boxes are ticked. To use the auto-test for thresholds, go to Image -> Adjust -> Auto Threshold. You can also automatically test the range of threshold algorithms on your image without scrolling through the dropdown list one at a time. ( Note: there will be more details on masks and binaries in the next sub-section). When you are happy with the selection (ie: the red covers the parts of the image you want selected well, without any additional background), close the threshold box to keep is as a threshold only (A in the image below), or click Apply (B in the image below) then close the threshold box to convert it to a mask. Reset will remove any overlayed threshold and allow you to begin again. Rather than manually adjust, you can also select your algorithm and use the Auto button to allow the program to adjust the threshold levels for you. The red box on the histogram shows which parts of the histogram are being thresholded. The top slider sets the bottom range of the threshold and the bottom slider adjusts the top range of the threshold. Choose the threshold that looks liek the best fit for your data then further adjust by moving the sliders above. The drop down box on the left (the one that says 'Default' in the image above) has 16 different auto threshold algorithms to choose from. In the window that opens, ensure the box beside Dark background is checked for fluorescence images. To apply a threshold, select your image and go to Image -> Adjust -> Threshold (or shortcut Ctrl+Shift+T).Ī red overlay will be placed on the parts of the image that are selected by the threshold and a window will open where you can adjust the threshold on the image. Threshold Imageįor a number of basic measurements an image first needs to have a threshold applied to it. The image Nuclei-1.tif and the image set ‘ RGBstackProjection, RGB-blue, RGB-green and RGB-red’ are used for demonstration purposes in this section. This section outlines the various options for masks and filters and how to apply them to your images prior to analysis. Creating thresholds and masks and applying filters forms the basis for a number of analysis methods.
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