Short manual - 2013/07/03

Growth increment measurements of the squid gladius using ImageJ software D. Vijai and Y. Sakurai
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan.

Contents
1 Spatial calibration of image and adding scale bar 1.1 1.2 2 3 Setting the scale Adding scale bar

Setting up the 3D Distance Tool Step-by-step recipe for counting growth increments 3.1 3.2 Make montage 3D Distance Tool

Optional method (without using 3D Distance Tool)

Spatial calibration of image and adding scale bar Spatial calibration measurements of images are calculated automatically by the ImageJ software and the process involves the following steps. Place a micrometer slide (slide with a finely divided scale marked on the surface) on the microscope stage. Magnify the image to a desired level (e.g. 20x), then capture the image and save it as calibration image in any raster format (e.g. png and jpeg). Now your image has got a scale (Figure 1-2), with a known length, which will be used for spatial calibration. 1

Figure 1-1: ImageJ Window

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Figure 1-2: Calibration image

1.1

Setting the scale a. Start ImageJ (Figure 1-1). b. Open the calibration image (Figure 1-2). c. Use the straight line selection tool from the tool bar to select a known distance on the image (holding shift key while drawing will give a perfect straight line). d. Then choose Analyze Set Scale (Figure 1-3). e. Enter the Known Distance (e.g. 4). f. Enter the Unit of Length (e.g. cm or mm). g. Check Global to apply to all images in the current ImageJ session. h. Click OK.

Figure 1-3: Set Scale Window

1.2

Adding scale bar a. Choose Analyze Tools Scale Bar (Figure 1-4). b. Specify the width, height (thickness), font size, colour and location c. Click OK. d. Use the Text tool in tool bar to write on the image (e.g. 20x). Page 2 of 9

Short manual - 2013/07/03 e. Use Edit Draw to permanently draw the text on the image. f. Save the image as calibrated image.jpeg (Figure 1-5).

Figure 1-4: Scale Bar Window

Figure 1-5: Calibrated image

Now you have a spatially calibrated image, which you can use to calibrate your actual gladius images, captured under same microscopic conditions. If you intend to analyse your gladius under various magnifications, make sure to capture separate calibration images for each magnification with the micrometer slide. Setting up the 3D Distance Tool The growth increments are measured using the ImageJ tool 3D Distance Tool. This tool can simultaneously count and measure growth increments. You can open the 3D Distance Tool with the following steps. 2 a. Choose Plugins Macros Install.

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Short manual - 2013/07/03 b. On the opened folder structure, select the ImageJ folder (Usually found in C:Program Files). c. Open the folders ImageJ macros tools. d. Select 3D-Distance-Tool.

Figure 2-1: 3D Distance Tool button

You will now get an icon (2 dots) in the tool bar (Figure 2-1). To avoid doing this every time we open ImageJ, we have to bring the 3D Distance Tool permanently into the ImageJ tool bar. This is done by following the steps below. a. b. c. d. e. f. g. h. 3 Open the ImageJ folder (Usually found in C:Program Files). Open the folders ImageJ macros tools. Copy 3D-Distance-Tool.txt. Open the folders ImageJ plugins Tools. Paste 3D-Distance-Tool.txt. Rename with underscores (3D_Distance_Tool.txt). Restart ImageJ. You will find it as a menu item under More Tools (>>).

Step-by-step recipe for counting growth increments For explaining the increment measurements using 3D Distance Tool we use a set of 6 images. These images are successive images of a single squid gladius captured in parts from the anterior end. Number of increments on each images are as follows; 8, 3, 5, 3, 1 and 4 (for explanation purpose revealed beforehand). 3.1 Make montage a. Load the calibrated image.jpeg and close it after setting the scale (follow the steps explained under 1.1. Setting the scale). b. Load the gladius images (6 images in our example) captured under same microscopic conditions (of calibrated image), by drag and drop into the same ImageJ session. c. Choose Image Stacks Images to Stack (If you drag and drop a folder, ImageJ can open all images directly as stack). d. Click OK (Stack is formed). e. Choose Image Stacks Make Montage (Figure 3-1). f. Enter Columns value as 3 and Rows as 2. g. Enter the Scale Factor value as 1(to retain the original scale of each image). h. Enter the Border Width as 10 (to append white space between images). i. Click OK.

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Figure 3-1: Make Montage Window

Figure 3-2: Montage of 6 images arranged in 3 columns

Now you will get a montage (combination of images), of 6 images arranged in 3 columns and 2 rows (Figure 3-2). If the images are named in an ordered way, the montage will have successive images automatically arranged. j. Choose More Tools (>>) Magic Montage. This installs new menu items in the custom tools area (Figure 3-3). Among the newly appeared menu items, k. Choose Montage Shuffler Tool to reorganize the images in the order starting from the anterior end of the gladius (if necessary).

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Short manual - 2013/07/03 l. Choose Annot Tool to label the individual images (Double-click Annot tool for more options). m. Choose bar to add scale bar (can add more than one scale bar).

Figure 3-3: Magic Montage buttons

3.2

3D Distance Tool Choose More Tools (>>) 3D Distance Tool. You will now get an icon (2 dots) in the tool bar. Double-click the icon. Check Draw Labels (Figure 3-4). Click on the first increment visible on the first image in the montage (increment count usually starts from the anterior end of the gladius). e. A reading pops up in a separate window. Safely ignore it, but do not close. f. Click on all successive increments (8 increments on our example) on first image in the montage, ImageJ marks the increment counts and measures the increment distance automatically. The results are displayed in a separate window. In our example our last count on first image is 8 (Figure 3-5; Figure 3-6). g. Before starting with the second image in the montage, you have to delete the first measurement shown in the pop-up table (Figure 3-6). This can be done by rightclicking on the measurement and clear. This is done for two reasons; first, that reading is not the actual increment width. Second, this will enable numbering the second image starting from the last count on the first image (in our example count on first image ends at 8, and count on second image should begin at same 8). h. Repeat the same counting process with second image. In our example we have 3 increments (8 to 11) in the second image. i. Before moving to third image, delete the first measurement of the second image (8th measurement) (Figure 3-7; Figure 3-8). This is because that reading is the inter-image distance and not the increment width. This will also enable to mark counting from 11 on the third image (i.e. count on second image ends at 11, and count on third image should begin at same 11). j. Repeat the above steps with all images till all visible increments are counted. In our example we got 25 counts and 24 increment widths (Figure 3-9). k. To export the data from ImageJ to MS Excel, Edit Select All; Edit Copy. l. Open MS Excel; Paste. a. b. c. d.

These measurements can be taken in duplicates or triplicates and averaged to minimise the measurement error.

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Figure 3-4: 3D Distance Tools Window

Figure 3-5: Growth increments marked and Scale bars added

Figure 3-6: Results Window for first image (Image A). Delete first reading

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Figure 3-7: Delete 8th reading

Figure 3-8: After deleting 8th reading

Figure 3-9: Final result

Optional method (without using 3D Distance Tool) a. Make montage (same as previously described in 3.1. Make Montage). b. Use the straight line selection tool from the tool bar to draw line between the increments.

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Short manual - 2013/07/03 c. After placing straight line between the increments, press t (or Ctrl+T) and b (or Ctrl+B) successively. t is to add the straight line to ROI Manager (ROI = Region of interest; Figure 4-1) and b is for making the line visible, while adding more lines. d. For the same options on the menu bar; Edit Selection Add to Manager (for t), and Image Overlay Add Selection (for b). e. Choose the Measure button on the ROI Manager (new window; Figure 4-1), to get the increment widths in a new window. f. Save or copy-paste the results to MS Excel.

Figure 4-1: ROI Manager Window

List of figures Figure 1-1: ImageJ Window....................................................................................................... 1 Figure 1-2: Calibration image .................................................................................................... 2 Figure 1-3: Set Scale Window ................................................................................................... 2 Figure 1-4: Scale Bar Window................................................................................................... 3 Figure 1-5: Calibrated image ..................................................................................................... 3 Figure 2-1: 3D Distance Tool button ......................................................................................... 4 Figure 3-1: Make Montage Window .......................................................................................... 5 Figure 3-2: Montage of 6 images arranged in 3 columns .......................................................... 5 Figure 3-3: Magic Montage buttons .......................................................................................... 6 Figure 3-4: 3D Distance Tools Window .................................................................................... 7 Figure 3-5: Growth increments marked and Scale bars added .................................................. 7 Figure 3-6: Results Window for first image (Image A). Delete first reading ............................ 7 Figure 3-7: Delete 8th reading ................................................................................................... 8 Figure 3-8: After deleting 8th reading ....................................................................................... 8 Figure 3-9: Final result............................................................................................................... 8 Figure 4-1: ROI Manager Window ............................................................................................ 9

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