DiameterJ Installation and User

Instructions
Installation:
Download and install ImageJ 1.48 or newer or Fiji (latest version). If you installed imageJ
before the end of 2013, you should uninstall your current version of ImageJ (DO NOT UPDATE)
and reinstall ImageJ 1.48 or newer. Before uninstalling, be sure to copy all of your old plugins
into a separate folder as these will be removed when you uninstall your old version of ImageJ.

Download the DiameterJ plugin h ttp://fiji.sc/DiameterJ#Download_Link, unzip the files and move
or copy the three folders into the plugins folder of ImageJ/FIJI.

If you do not know where the plugin folders is located, navigate to the location where ImageJ is
installed and find the plugins folder. If defaults are chosen, you will find the plugins folder in
one of the locations below:

Windows - C:\Program Files\ImageJ\plugins or C:\Program Files (x86)\ImageJ\plugins

OSX - /Applications/ImageJ/plugins
Linux - directory as specified by user

Once downloaded, simply move the unzipped DiameterJ file(s) into the ImageJ/FIJI plugins
folder and restart ImageJ.

For more questions, please see http://imagej.net/Plugins.

Image Segmentation:
If your image is not a binary image (only black and white pixels), complete the
image segmentation steps below before performing DiameterJ analysis:

Image segmentation is the process of converting an image into a binary representation of your
image, where the white pixels represent the portions of the original image that you would like to
measure and the black pixels represent the background that you would not like to measure.

1. Open ImageJ
2. Go to: Plugins-->DiameterJ-- DiameterJ Segment to segment images.
3. A pop-up window will appear asking for several pieces of information
4. First enter in the dimensions of your image in pixels. These must be correct for accurate
image cropping and segmentation.
5. Next select whether your image needs to be cropped and, if so, what the pixel
coordinates of the box you would like to create are.
a. In general, these coordinates define the top left corner of the box you would like
to analyze and the bottom right corner of the box. For now DiameterJ only
supports square/rectangular cropped regions of interest, however, in the future
we hope to support dynamic and free-hand regions of interest for cropping.
6. DiameterJ Segment can use up to 24 different segmentation algorithms to segment your
image. Each of the check boxes under Segmentation Algorithms to Use represent 8 of
these algorithms. Select as many or as few as you want. However, in order to convert
your image to a black and white image that is able to be analyzed later at least one box
must be checked and None cannot be checked.
7. Batch processing is enabled by default but if the user only would like to analyze one
image select the No radio button.
a. NOTE: If the user only wants to analyze one image that image must be opened in
DiameterJ prior to starting the DiameterJ Segment or DiameterJ 1.XXX plugin.
8. Click the Okay button after you have made all of the selections you would like to make.
9. A pop-up window will appear asking what folder you would like to analyze images from if
you chose to batch analyze images, or asking where you would like to save the output of
the analysis of the image that is open in ImageJ if no batch analysis was selected.
a. If batch was selected output files will be saved in the same location that the
images the user selected are in

10. The code will then produce three new folders within the folder where the image that you
selected for analysis is located. One folder is called Best Segmentation, another is
called Montage Images, and the third is called Segmented Images.
a. The Best Segmentation folder contains no images.
b. The Montage Images contains a montage comparison image of the original
image to the 8, 16, or 24 different segmentation methodologies. Look at each of
the montage images and find the name of the image with the most accurate
representation of your fibers. (Names of the images shown in the montage are
on the bottom of each image in Red.)
c. The Segmented Images folder has all of the segmented images from the
montage images. Copy and paste the images you have determined to have the
best segmentation into the Best Segmentation folder.
 11. Visually inspect these images to make sure that the segmentation worked well and that
the segmentation represents the image you want to analyze. If it does not, try the other
segmentation algorithms.
12. If none of the 124 algorithms segment your image well, you can take the best
segmentation results from running the algorithm and manually correct any remaining
errors until the images accurately reproduce the fibers, you can manually create your
own segmentation, or you can use your own segmentation algorithm to create the black
and white images. DiameterJ only needs a black and white (binary) image to analyze
and thus it doesnt matter how that image is created.
13. After copying the best image into the Best Segmentation folder, the user can delete all
other unused images/montage images.
a. This step is not required it just saves a lot of hard drive space

DiameterJ Analysis:
Analyzed images MUST be white fibers on a black background. If you have black
fibers on a white background, invert the image to analyze it with DiameterJ.

In ImageJ, go to:

Plugins-->DiameterJ-->DiameterJ 1-XXX to analyze the segmented image.

1. A pop-up menu will appear that gives the user several options for analysis
2. First choose what type of orientation analysis (if any) you would like to perform on your
images.
a. OrientationJ is the default because it is fast and provides a good comprehensive
graph of fiber orientation. However, it will not provide raw data for the user.
b. If the user requires the raw data for the orientation graph, they will have to either
run OrientationJ manually, OR use FIJI and select the Directionality plugin for
analysis.
c. The Directionality plugin only works for FIJI, is very slow, and leaves windows
open that cannot be closed in any way but manually.
3. Next, the user must decide if they want DiameterJ to report all outputs in pixels (default if
no unit conversion is selected) or if they want to convert pixels into real units such as
micrometers or nanometers
a. If the user would like DiameterJ to convert units they will need to know the
distance to pixel transformation. The protocol for how to perform this procedure
can be read in detail below.
4. If the user would like to know where a particular radius of fiber is occurring (perhaps in a
previous run the user saw a peak where they did not expect one or there is high noise in
a radius histogram) the user has the option for DiameterJ to create a separate graphic
showing the location of radii in an image specified in the range specified by the user.
a. The user must select the Yes radio button under Identify Specific Radius
Location heading and enter in the lower and upper bounds of the radii they
would like to see for this option to work.
5. Batch processing is enabled by default but if the user only would like to analyze one
image select the No radio button under the Batch Processing heading.
a. NOTE: If the user only wants to analyze one image that image must be opened in
DiameterJ prior to starting the DiameterJ Segment or DiameterJ 1.XXX plugin.
6. Additionally, DiameterJ can now combine the analysis of multiple files into single
summary sheets if the Yes radio button is selected under the question Do you want
DiameterJ to combine analysis from all images?
a. Note this combines all images analyzed into summary files. Thus, if multiple
samples are contained in the same folder and DiameterJ is analyzing that folder
it will combine the analysis from multiple samples into the same summary files.
7. Click the OK button after you have made all of the selections you would like to make.
8. A pop-up window will appear asking what folder you would like to analyze images from if
you chose to batch analyze images, or asking where you would like to save the output of
the analysis of the image that is open in ImageJ if no batch analysis was selected.
a. If batch was selected output files will be saved in the same location that the
images the user selected are in
9. DiameterJ will then analyze all images in the specified directory and will separate its
analysis into three folders: Diameter Analysis Images, Histograms, and Summaries.
These folders will be located in the directory where the analyzed image is located. The
file labeled DiameterJ Output Description describes what the outputs are in each of
these folders.
Pixel to Unit Distance Transformation:

All measurements given by DiameterJ are in pixels by default.

1. To determine the pixel to unit distance (nanometer/micrometer/millimeter) conversion,

there are two options depending on how much metadata your imaging software stores
with your image. It is possible that you already have the pixel to distance conversions in
the metadata and thus getting the distance transformation number is easy. Simply go to:
a. Analyze-->Set Scale

2. Look at what the unit of length is. If its inches, then your SEM doesnt store the
necessary metadata information with the micrograph and youll have to do the
conversion by hand.
3. In the case that you have to calculate by hand, open any micrograph (at the
magnification that you have taken your SEM images) in ImageJ.
4. In ImageJ go to menu:
a. Analyze-->Set Scale
5. Select Click to Remove Scale
6. Close the scale window
7. Place your mouse over the scale bar and hit the + key to zoom-in.
8. Continue to zoom-in on the scale bar until it fills the screen.
a. After zooming in, you can widen the default width of the window that ImageJ
chooses just like any normal window.
9. Next, select the Rectangular tool from the menu bar.
a. If the rectangular tool isn't available, go to the tile that has >> and click Restore
startup tools from the drop down menu.
10. Next, draw a square or a line that spans the scale bar.
11. To get the length or width of this rectangle, simply look under the toolbar of ImageJ as
you are drawing the rectangle.
a. As soon as you stop drawing the line, the measurements will disappear, so dont
let go of the cursor until youve seen and noted the rectangle width.

12. Record the number that w" is equal to.

13. Then its simply a matter of entering in the length of the scale bar in real units (10
microns above) and the number of pixels you just recorded (w) into the Diameterj
1.XXX menu and selecting Yes to unit converion.
14. All units in all output files will then be in terms of microns, microns2, etc.