Size Calibration and Photographing

A version of the TEM filter image, rendered for publication with a micrometer marker and with a black background, is shown in Fig. 15. This figure was made using the calibration feature of NIH Image, the Text tool, the line drawing tool, and the Photo Mode command.


Figure 15-- Contrast enhanced version of the Filter image suitable for publication, with micrometer bar. Black background not shown. (The figures were made by 'capturing' the screen (shift-command-3), a function provided by the Macintosh operating system, or by pieces of software such as 'Capture'*. Capturing does not work in the photo mode, so only the image window is shown in this figure.)
Get a version of the image with suitable contrast using the Revert to Saved command, the Process - Enhance Contrast command, and the Process - Apply LUT command.

The image field of view is 15 m wide. Click on the line selection tool. Make a line selection across the entire width of the image. Invoke the Analyze - Set Scale command. Select Micrometers for the units, then type in 15 for the known distance. Click 'OK'.

Click on the widest line in the line width tool. Then click on the Draw Lines tool (just below the paintbrush). Draw a horizontal line with the mouse in an uncluttered area of the image, keeping an eye on the length in the Info window. When the line is as close as possible to 1.0 m long, release the mouse button and the rubber band line will be replaced with a fat line or rectangle for the marker. If this line is not the correct color, IMMEDIATELY use the Edit - Undo Editing command to erase the line. Select the appropriate color as indicated by the color of the paintbrush (see above), and draw the line again. (Note 2. This could also be done by creating a 1 Ám wide rectangular selection and using Edit - Fill .)

Next, click on the Text tool ('A' in the Tool window), and click this tool to the right of the micrometer bar. Any time up to and including this point, use the Options - Size - 18 command and the Options - Style - Bold command, to make the type suitably large on the image. Then type "1 option-m m " to write "1 m" directly on the image to annotate it. Use the File - Save As... command to save this file under a different name, if desired, as this has permanently modified the image.

Select a background color by clicking on the erase tool, then clicking inside the LUT window - where the erase tool has temporarily changed to the eye dropper tool (the paintbrush tool will work in a similar fashion, but it is the color of the eraser that affects the background color.) The color of the eraser will change to the color clicked on with the eye dropper tool. Black was selected for this figure. Move the image to the desired position on the screen by dragging the title bar of the image window. Invoke the Special - Photo Mode command. The entire screen will turn black, except for the image, which appears without the title bar. The screen can be photographed directly, or using a slide copy camera. Alternatively, the image can be cut and pasted into a larger blank window of the desired color using the File - New command - the new window will have the color of the eraser. This larger window can be printed using the File - Print Image command.

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