Measuring Optical Density with AIS Using 16-Bit Images

1. Obtain a 16-bit image of your sample using your favorite program. Save it in the same format that the calibration image is in; if these formats are different, then any measurements made will be meaningless. Make sure that the sample 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 with 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 sample file 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 under the menu bar and click on the calibration icon. It is the icon farthest on the left and has a picture of a ruler on it.
8. Go to the menu bar, click on File, and select Open. This will bring up the Open window. Select the appropriate calibration (the appropriate calibration is the one obtained under the same set of conditions as the sample) from the box on the right of this window and click on the OK button. AIS will bring up a warning window saying that the calibrations need to be redone. Click on OK. This warning is just a reminder that images taken under different circumstances than the standard image will require a different calibration than the loaded calibration. The calibration OPTICALDENSITY.CA is a 16-bit TIFF image obtained with the Leaf Scanner; if the sample image is also a 16-bit TIFF image obtained with the Leaf Scanner, then this is the desired calibration.
9. After the calibration has been loaded, go to the icon bar, and click on the sample icon (the second icon from the left).
10. There should now be a window with the sample image on the right of the screen, two columns labeled Target and IOD on the left 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 top 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.
11. Go to the image of the sample and center the red box on the image at a point where an optical density measurement is desired. Click on the left mouse button. This should make numbers appear in the two columns at the left of the screen. The number in the Target column corresponds to the number of the measurement. For instance, the number 1 means that this is the first measurement made. The number in the IOD column corresponds to the average optical density of the entire area enclosed in the red box at the time of measurement.
12. Repeat Steps 10 and 11 until all optical density measurements have been made.
13. Close AIS.

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