Creating 16-Bit Optical Density Calibration Files with AIS

1. Obtain an image of the optical density standard using your favorite program. Save it in the same format that the sample images will be in; if these formats are different, then any calibrations made will be meaningless. Make sure that these files are in a PC-compatible format, because the AIS program is on a computer that can not read Macintosh files.
2. Open AIS. It is in the Start menu under Programs.
3. Go to the icon bar just below the menu bar and click on the sample icon. It is the icon second from the left and has a picture of a black cross and brown dots on it.
4. Now go to the menu bar, click on Settings, and select Display format. This will bring up a window with several options for the display. Click on the white button next to the words 16 bits and then click the OK button.
5. Go back to the menu bar, click on Image, and select Retrieve. This will bring up a window titled Retrieve Image. In this window, select the correct format and the desired optical density standard image and then click the OK button.
6. There should now be a window titled Image View—Ch 1 with the image selected in Step 5. If there is not, go back to Step 2 and try again.
7. When the image has loaded, go to the icon bar just below the menu bar and click on the calibration icon. It is the icon farthest to the left and has a picture of a ruler on it.
8. Go to the menu bar, click on Establish, and select Density. This should bring up a row of buttons just below the icon bar. Click on the button next to the words Cal stds.
9. A window titled Open Density Standards File will appear. In this window, name the calibration to be made and then click on the Create button.
10. This will bring up another window that allows the entry of values. It is where the actual optical density values of the standard image should be entered. Enter one value per line and enter them in order of increasing optical density. Click on the Done button when finished.
11. The program will ask if the file should be saved. Tell it to do so. It will then switch to the Open Density Standards File window. Make sure the new calibration is selected and click on the OK button.
12. There should now be a list of the calibration values from Step 10 on the left of the screen, a window with the optical density standard image on the right of the screen, and a window titled Sample at the bottom left of the screen. Go to the Sample window and double click on the second button from the left in the first row. (It should have a picture of a box with a dot in its center on it.) This will bring up a window titled Rotatable Box Cursor Settings. This allows the width and height of the measuring tool to be changed. Adjust the values for width, height, and rotation until they are satisfactory, and then click OK.
13. Go to the image window on the right and center the square mouse pointer on the area of the image corresponding to the first optical density value on the table at the left. Click the left mouse button. This should make an AIS value appear next to the first optical density value in the list. Next, go to the region of the image corresponding to the second optical density value on the table at the left. Click on the left mouse button when the pointer is centered on the appropriate area. Again, an AIS value should appear next to the second optical density value in the list. Repeat this procedure until all of the optical density measurements have AIS values next to them. Please note that these AIS values correspond to the entire region within the square pointer, not just the center. When you have finished, go to the menu bar, click on File, name the new calibration, and select Save.

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