Calibrating Length Measurements Using TEM Images II

TEM Images of Beef Liver Catalase Crystals

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

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 selected and a toolbar. At the top right of the toolbar there is a picture of a rectangle with dotted lines (the marquee tool). Click on this picture.
3. By holding down the Shift key while clicking and dragging the mouse, select an area of the TEM image in a square-shaped box. Make this square with side equal to a power of two. The width and height of the square are displayed at the top of the Info window while the square is being drawn. If the Info window is not present, go to the menu bar, click Windows, and select Info. Remember the size of the square used in this step.
4. By clicking and dragging the mouse inside the square, move the square so that it encompasses the part of the TEM image desired. The part of the image inside the square will be used to compute the Fourier transform mentioned in the next step, so be sure that the square is in the right place.
5. One the square has been selected and positioned, go to the menu bar, click on Process, and select FFT. This will produce a new window containing an image of the Fast Fourier Transform of the TEM image inside the square.
6. 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.)
7. By clicking and dragging the mouse, draw a line connecting the dots (amplitude maxima) in the image. (See the figure at the right.) Make the line as long as is easily possible.
8. Measure the length of the line in amplitude maxima 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 amplitude maxima because of the reduction in estimation errors.)
9. 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.
10. Now go back to the menu bar and 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.
11. 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 7. Write down or remember this number.
12. A calculator will be needed for this step. To find the pixel length, use the following formula.

    

    where pl is the length of a single pixel in nm, size is the length of the side of the square selected in Step 3, pix is the length of the line from Step 7 in pixels, and #am is the length of the same line in amplitude maxima. The factor of 20.6 comes from the 20.6 nm spacing between adjacent atoms in the catalase crystal, so this factor must be changed if a different type of crystal is used.

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