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Alignment Using SEM Align

by JCF, V. Hui, KMH

1. Choose the middle section in the series as a reference point. This section will not be transformed.
2. Align the series incrementally. Select incremental for mode under the Transform:Align Points Menu.
   1. Align the upper half of the series.
      1. For example, if the middle section is section 50. Start by aligning section 51 to 50.
         1. Select corresponding points* in the two sections.
            1. Go to section 51.
            2. Select a cross-sectioned mitochondrion** that also appears on section 50. Click on it with the mouse to generate a colored spot in the center of the mitochondrion**.
            3. Go to section 50.
            4. Select the same mitochondrion** and put a corresponding point in its center.
            5. Go back to section 51 and repeat the process for 2 or more cross-sectioned objects. Use the '/' key to rapidly go back and forth between the 2 sections.

            *You should enter a pair of correspondence points together. Putting all the points on one section and then all the matching points on the other section will lead to problems if the ordering is mixed up. Also, if you delete a point on a section and then add a new one, the ordering will also change unless the deleted point was the last one entered on that section.

            **Microtubules can also be used if the images have sufficient magnification to make selecting microtubules feasible. Select round microtubule profiles in cross-sectioned dendrites or axons.

         2. Go to the upper section (51 in this case) and transform it using Linear from the Transform:Align Points Menu.
         3. Check the alignment using blend (Ctl-B) or flickering (hold the '/' key down).
         4. If alignment is not good enough, add corresponding points to the regions where it is misaligned, reapply Linear, and check it again.

            Always try using Linear first. Use Deformal or Quadratic only as a last resort.

      2. Adjust for optimal contrast. Try to match contrast on all sections.
      3. Save the Transform for section 51 by selecting Save from the Transform Menu when on Section 51.
      4. Clear the Transform for section 51. This is necessary for incremental alignments only.
      5. Go on to the next higher section, e.g. 52 in this case, and align it to the previous section (51).
      6. Continue aligning higher sections to lower sections until reach the end of the series, i.e. 52 to 51, 53 to 52, etc.

   2. Align the bottom half of the series. Going downwards align each section to the one closest to the middle section, e.g. 49 to 50, 48 to 49, 47 to 48, etc.
   3. Fix the contrast of each section as you align. Uniform contrast will result in clearer blends between sections and make the series easier to work with when tracing.
   4. If you encounter a badly mangled section, i.e. with a large fold or crack in the middle of the section, skip it. Do not try to align it in incremental mode as it will distort the rest of the series. Leave the transform of this section cleared and instead align the adjacent sections to each other. For example, if 56 has a fold, but 55 and 57 are ok, align 57 to 55. Later, in Absolute mode, align 56 to 55 or 57 as best as possible.

3. Copy the incremental transforms (all files with the '#' as the first character) into a separate folder, so you can go back to them later if necessary.
4. Change the alignment to absolute. This can be done by selecting Incremental->Absolute from the Series Menu. This needs to be done twice for the series, once for the lower portion of the series to the middle section and then again from the middle section to the last section. Indicate the order of alignment by selecting the Ascending or Descending item.
5. In absolute mode you should be able to see if your sections are aligned without clearing the previous section. Starting with section 1, page through each section to see they match up properly with adjacent sections in Blend mode.
6. If the sections in absolute mode are not well aligned you will need to make changes. The only way to change individual sections without affecting the whole series is to make changes in incremental mode. You can get back to incremental by using Restore from the Series:Transforms Menu, or by copying the incremental transforms back into the series folder.

   It is not advised to use the Absolute->Incremental conversion at this point. These conversions (Absolute->Incremental and Incremental->Absolute) are approximate numerical conversions and repeated use can buildup numerical errors.

   You can tell if a series is in incremental by the fact that only when the section closest to the middle section is cleared will it look aligned to the next section.

7. Use Apply to Images from the Series:Transforms Menu to generate a new aligned series from the final absolute alignments.
   1. (Optional) Select Type In from the Transforms Menu. Set the Keystroke column of the X and Y Translations to 10. (This gives the arrow keys a movement of 10 pixels each.) Select OK.
   2. Enter View Mode by hitting the space bar. This allows you to adjust the View Transform and prevents you modifying section transforms with keystrokes.
   3. Starting a section 1, use the Arrow keys to bring the bottom and left edges of the section into view. Go through the entire series making sure that the lower and left edges are visible on each section. The idea is to get each section to come into view so it will also be in view when Apply to Images is done. This operation can be done in blend mode and therefore can be combined with checking the absolute alignments.
   4. (Optional) Incorporate the View Transform into the section transforms by selecting Transforms - Adjust By - View Transform from the Series menu. After this, select Clear Offsets from the View menu. This should give you the same view as before, but with the offsets permanently combined with the absolute transforms. Thus, you can return to this view later if necessary.
   5. Select Apply to Images to bring up the Apply to Images dialog. The width and height will be set to capture the upper and right edges of all sections in the series. Write these numbers down for later reference.
   6. Carry out the Apply to Images operation on one or two sections. Check the result (the new series images) in IGL Trace or SEM Align.
   7. Once acceptable parameters have been determined and tested, use Apply to Images on all of the sections, including section 0.

Last Updated: 04/30/03