Calibrating Length Measurements Using TEM Images I

TEM Images of Diffraction Grating Replicas

Please note that all pixel lengths are magnification-dependent. That is, the pixel length calculated will be different for different magnifications.

1. Open NIH Image.
2. Go to the File menu, choose Import, select the TEM image acquired above, and click on Open. This should bring up the TEM image and a toolbar. Go to the toolbar and select the line selection tool. (It is the button fourth from the top next to the paintbrush. It looks like a dotted line.)
3. By clicking and dragging the mouse, draw a line along one of the vertical lines of the squares in the image. (See the figure on the right.) Make the line as long as is easily possible.
4. Measure the length of the line in squares and write down or remember this number. (The most accurate calibrations are obtained with the length of the line equal to an integer number of squares because of the reduction in estimation errors.)
5. Go to the menu bar, click on Analyze, choose Options, and make sure the box next to Perimeter/Length is checked. If it is not checked, click on the box to check it.
6. Now go back to the menu bar, click on Analyze, but this time choose Measure. This should bring up a window titled Info. Sometimes the Info window is hidden behind other windows and so it is not visible. If this is the case, go to the menu bar, click on Windows, and select Info. This should make the Info window appear.
7. In the Info window, look for the number next to the word Length. This is the length in pixels of the line drawn in Step 3. Write down or remember this number.
8. This step will require a calculator. To find the pixel length, use the formula below:

   

   where pl is the length of a single pixel in nm, sq is the length of the line from Step 3 in squares, and pix is the length of the same line in pixels.

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