Finding Optical Density with the Leaf Scanner

Please note that the Leaf Scanner requires at least 2 hours of warm up time before it will produce reliable data. Ignore the sign on the scanner itself referring to a 15 minute warm up. The 15 minute warm up is only adequate for nonquantitative imaging.

1. Open Adobe Photoshop.
2. Go to the File menu, choose Import, and then select Leafscan 2.2 SCSI.
3. A window titled Leafscan 2.2 SCSI will appear. On the left side of this window there will be several buttons that control the scanner settings. The button at the top should have dimensions on it and maybe a label like Portrait or Landscape. Click on this button and select 4x5 in. Portrait.
4. The button directly below the top button should say either Color or B&W and Positive or Negative. Click on this button and select B&W Positive.
5. The button closest to the bottom should say More Settings.... Click on this button, select 16-bit color, and choose Standard Format (8-bits per pixel).
6. The third button from the top should say Calibrate. Click on this button. This will bring up a window saying that a saved calibration is available. Do not use the saved calibration. Click on Recalibrate instead. The scanner must be recalibrated for each scan.
7. Next, a window will appear asking to remove the film holder from the scanner. This will happen even if there is nothing in the scanner, but check to make sure that nothing is there anyway. If one of the metal holders is present, pull it out gently. Use a small amount of downward pressure to pull the film holder out of the scanner. After making sure the scanner is empty, click on the Continue button on the computer screen.
8. The scanner will then bring up various windows concerning its progress during the calibration. When it is done, it will return to the Leafscan 2.2 SCSI window.
9. When the scanner is finished calibrating, choose a film holder from the box at the right of the computer. Put the sample in the film holder and gently load the film holder into the scanner.
10. Go to the buttons on the left side of the window, find the one marked Focus, and click on it. Again, the scanner will display its progress.
11. Now go to the buttons on the left side of the window, find the one marked Prescan, and click on it.
12. When the scan is finished, an image of the sample will appear in the Leafscan 2.2 SCSI window. There should also be a black (white, if the image is dark) rectangle on top of the image. (The rectangle should not be blue.) If the black rectangle is not present, look for a row of buttons just to the right of the 4x5 in. Portrait button. Click on the one closest to the 4x5 in. Portrait button. It should have a picture of a black square with the lines extending past two of the corners on it. This should make the black rectangle appear on the image.
13. By clicking and dragging the sides of the rectangle, adjust the rectangle so that it encloses the area of the sample desired. If using Photoshop 5.0, first click on the little lock icons next to the words Height and Width in the window on the left of the screen. When the locks look as though they are unlocked, the size of the rectangle can be adjusted. Once size of the rectangle is adjusted, however, click on the locks again to lock them. The option to scan will not be available until the locks are locked.
14. At the left of the Leafscan 2.2 SCSI window there will be a button labeled Resolution. Click on this button and set the resolution to the value desired.
15. Click on the Final Scan button. Again, the scanner will display its progress.
16. There should now be an image of the area previously enclosed in the box. The image will be the one used to calculate the optical density, so make sure it is satisfactory. If the image is unsatisfactory, discard it and go back to Step 2.
17. If the image of the sample is satisfactory, go to the menu bar, click File, and select Save. Name the image, select the TIFF format, and click on the Save button.
18. Quit Adobe Photoshop. Now open NIH Image.
19. Go to the File menu, click on Open, select the image saved in step 10, and click on the Open button.
20. Now go to the menu bar, find Special, and choose Load Macros. This will bring up an untitled window. Click on the Desktop button on the right of the window, then choose DaVinci Workspace from the options at the left, select the folder cgreen, and double click on the macro Measure Optical Density.
21. There should be an image of the sample and a toolbar. At the top right of the toolbar there should be a button showing a box with dotted lines (the marquee tool). Click on this button.
22. By clicking and dragging the mouse in the image window, enclose an area of the sample. Go to the menu bar, click on Special, and select Measure Optical Density. This will bring up a small window called Info, if it is not already present.
23. In the small window, there will be several values corresponding to different properties of the pixel values in the box. Look for the number next to the word Mean OD. (Not the number next to Mean.) This Mean OD number is the average optical density of the area of the sample enclosed in the box in step 22.
24. Repeat steps 22 and 23 for other parts of the sample that require optical density measurements. Close NIH Image.

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