To generate a basic intensity measurement we apply a similar method as the basic area measurement. Compare this result to the previous 2 values. Repeat the measurement ( Analyze -> Measure). ROIs will apprear around all of the masked nuclei. (Masked area must be black on a white background here as the ROI selection will be for the black component of the image) Go to Edit -> Selection -> Create selection. You can fix this with a simple additional step. But often It will still measure the entire image, disregarding the selected/masked area. Sometimes FIJI will measure a mask as expected perfectly fine. Compare here the measruements from a Mask and a threshold on the same image. If you have used a mask for your measurement, you may notice a rather large number. This is because this method measures the mask or threshold as a single entity. You will notice there is only one measurement. This will give you a results table with the area of the nuclei. Now that the parameters are set, to measure the area of the nuclei using the parameters you have just set go to Analyze -> Measure (or shortcut Ctrl+M). Here we will turn on Area only.Įnsure you also have Limit to threshold selected in the options so that FIJI does not measure the entire image. In the Set Measurements window you can choose what you want to measure by clicking on or off the checkboxes beside different parameters. To measure the area in either your thresholded image or your mask, you first need to set your output parameters. You should now have a mask that nicely represents the nuclei in the original image. Find an option that works for your image. Set a pixel radius that captures all spots outside the nuclei, in my example the default setting of 2 works nicely. Turn on the preview, ensure it is set to act on the Bright or Dark part of the mask underthe Which outliers drop down menu (this will be dependant on your mask colours - for me it needs to be set to Dark). Go to Process -> Noise -> Remove Outliers. Next we will remove the small spots detected outside the nucleus using the Remove Outliers filter. This will complete the mask over areas that contained gaps previously. To take care of these 'gaps' in the nuclei mask we are going to use the Fill Holes option found under Process -> Binary -> Fill Holes. You can see below that we have some small speckled areas detected outside the nuclei, as well as some spots within the nuclei that are not masked completely. In this image mask there are a few blemishes that may affect the measurement. If you are working with a mask you can perfect your selection using filters. Which is why we often create a mask instead. If you choose to work with the threshold you can skip straight to measurements from here, however the threshold is not always a perfect selection of the data. For your own data you will need to find a threshold that works for you.Ĭlose the options box to keep the selection as a threshold, or click Apply to generate mask as previously described. Here I have used the Default algorithm set at 65 and 255. Then go to Image -> Adjust -> Threshold (or use short cut Ctl + Shift + T).įit your threshold to the data as best as possible. This image is already grey, but apply a grey LUT if you are working with a colour image and duplicate the image if you would like. You may also like to Duplicate the original image and work on a copy. Check that the scale is calibrated before carrying out any further steps.įor colour images I like to apply a grey LUT to allow better contrast between the stain and background during thresholding. In this example we will use a threshold, but you can also select a specific area to measure by using an ROI. Without a selection, the program would give an area measurement for the entire image, rather than just the area of interest. We also need to select an area within the image that we want to measure. Checking and setting the scale calibration was covered in FIJI Basics. To perform any measurements on our image we must ensure the scale is calibrated. In this section we use the images RGB-blue.tif, RGB-green.tif, NeuralTubeRed.tif and Nuclei-1.tif for demonstration. Here, our instructions become more speicified as wel go through several examples of common measurements using the different tools in FIJI. Many of the simple FIJI functions that you have previously learnt about can be combined with other tools for more complex processes to analyse and measure your data.
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