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Open ImageJ. You will see the following: 

Figure 3. ImageJ Graphic User Interface (GUI).

Start by opening one picture. Instructions for opening a whole folder are shown in video tutorial on the Website. To open an image, FILE>OPEN>{IMAGEFILENAME.TIF} If you downloaded the example files, a good starter is found in the ‘40 Micron VMD’ folder. Use ‘4-2-M-T_0001’. Usually it is best to start with a blank, but for the first example it’s better to use a card with a good number of stains. The file will open in a separate window.


Next, set up the parameters that will apply to the image by going to Analyze>Set Measurements. Figure 4 below shows the resulting ‘Set Measurements’ menu. Once the boxes are checked they will remain checked through the rest of the session until they are changed by the user. Select all the boxes checked here:


Figure 4. Image file opened, Analyze>Set Measurements menu displayed.

Next, go to Analyze>Set Scale. This will tell ImageJ what a pixel value is in microns. At 4800 DPI each pixel is 5.29 microns. This could also be set as millimeters or centimeters. For this example Microns will be used. Again, once these are set they will remain through the session. If ImageJ is closed they will need to be re-set.


Figure 5. Set Scale. One pixel equals 5.29 microns for a 4800 DPI scan. 

The next step is the most interesting and important. Thresholding the picture tells ImageJ what in the image is a particle, and what to ignore. There is a more detailed video on the website explaining this. It is a good idea to experiment with this a little with each new set of scans. To Threshold, go to Image>Adjust>Threshold. Use the values shown next to the slider bars below along with the example image. Write these down for use with future images.


Figure 6: Image prior to thresholding, example threshold values shown at right.

After setting the slider bars to the values shown in the image above, click the ‘Filtered’ button and you will see the image transform in preparation for particle analysis.

Figure 7. Image after thresholding. Note intensity of droplets that were not previously visible.

Each red dot will be measured by ImageJ. In this scenario, all non-magenta colors outside the range specified in "Hue" have been excluded, eliminating most of the potential dirt, dust, and contaminants. As a side note, when you zoom in you will see a lot of random pixels and incomplete drops. These are all spray tracer. By running a Blank Calibration first, you can be confident that if a pixel has turned red in the thresholding, there was dye stain in it. Some pixels may have had a stain so faint that the scanner did not detect it; this is evident in the donut shaped stains.

Now run the Particle Analysis. Select Analyze>Particle Analysis and the Screen in Figure 8 will appear. Set the minimum size to 28 microns square. This is just over the size of 1 square pixel, so any stain of 2 pixels or more will be measured. There is more discussion on this in the website. Choose Display Results for detail on each stain, Summarize for summary statistics, and Include holes to measure the areas inside stains that might not have registered in the thresholding. Circularity of 0-1 means that no matter how misshapen a droplet, it will be counted. Once this is done, hit OK.


Figure 8. The Analyze Particles window.

About this window:

Size(micron^2): will allow you to limit stains to a certain minimum size. Since its based on area, if you want to only measure stains larger than 1 pixel, set the range to 28-Infinity. Assuming your image was 4800 DPI, 5.29 microns per pixel squared equals 27.98 square microns, therefore any stain 28 square microns or larger is at least 2 pixels. Often, as your percent area cover gets greater, you will see there is an increasing amount of what appears to be "noise" at the edges of the droplets. It's unclear whether this is reflection in the scanner due to the higher intensity of color in the target range that causes false positive color perception, or if something else is going on. There is a little bit of art in this that is explained more in the video on thresholding. 

Notes: There are some (very general non-absolute) rules of thumb for setting this range. You may find these useful later. 

• Very low coverage (less than 2%): 0-infinity is the place to start.  
• 2-5% coverage: Read stains greater than 1 pixel (28 microns^2) or larger. 
• 5-15% coverage: read stains 4 pixels (111.93 microns^2) or larger. 

Circularity: Leave this at 0-1. This will count all droplet stains no matter how misshapen.

Show: Begin with the default, nothing. This command allows you to also create a graphical output for the image along with the tabular data. For beginning, leave this blank to avoid confusion. Once comfortable with the basic routine, Experiment with the different output options. An excellent technical description can be found in the Manual, section 30.2, on page 133.

Check Boxes: 

Display Results (checked): creates a table of each particle found and its properties that you have specified in the "set measurements" routine above. Each subsequent image's data will be added to this table.  

Clear results (NOT checked): will delete the data from your prior image scan. Do not check this box!

Summarize (checked): will create a separate table from the Results table  with summary data for the entire image (area covered, number of hits, etc). Each subsequent image's data will be added to this table.  

Add to Manager (unchecked)

Exclude on edges (unchecked): If checked, removes any stain touching the boundary of the image. Since the primary use of this analysis is percent area covered and hits per area, this should not be selected, as it will reduce the value of both. 

Include holes (checked): Sometimes droplets make circular stains with holes in the middle. Checking this box will fill that "donut hole" area in the droplet size and percent area covered calculations.

Record Starts (unchecked): output not used in this type of analysis.

in situ Show (unchecked): Also not used in this analysis routine.