Neurons in Serial Sections—3: Align Section & Tracing
Neuronal processes have been traced in the first section.
Move to the subsequent section and align tissue and tracing from the first section:
Assumption: The current section lies next to the previously traced section.
- At low power, align the region in this section with the previous tracing. See Aligning Serial Sections.
- This preliminary alignment will facilitate the upcoming alignment at higher power.
- Trace anatomical regions that will act as fiducial points.
- Select a higher power.
- Focus on the top or bottom of the new section and locate fragments (stumps) of the processes. The fragments now in focus should be reasonably close to the low endings of the previous section. See Tracing Trees: Ending Types.
- To show ending labels, select use the Options>Display Preferences>Neurons tab and check Show Ending Labels.
- Fine-tune the alignment at high power:
- Focus on the stumps in the new section. These fragments are the continuations of the processes to which you applied low endings in the previous section.
- Navigate around your tracing identifying which low endings of the tracing easily match the stained high endings in the current section.
- Count the pairs of physical endings and traced low endings that match well. Use a slightly lower magnification in order to see more endings in a single field of view.
- Use Tools>Match to align section and previous tracing.
- In the Number of match points field, enter the number of pairs that you have just counted.
- To perform the alignment without completing all match points, right-click and select Accept As Is.
- If the tracing is not satisfactorily aligned with the current section, repeat Tools>Match.
- Set the correct Z value of the new section. This value should correspond to the value in the Z meter.
- Click Edit>Select Objects.
- Hover over each low ending (indicated by the letter L). A pop-up box displays the X, Y and Z values. The Z values should be reasonably close to the values assigned to the top or bottom of this section.
Differences in mounting of sections may cause distortion of the tissue that adversely affect alignment. The goal of this procedure is to achieve the best possible match, which is rarely a perfect match between all endings. There will inevitably be instances when you will trace despite the absence of stumps, and instances of overlapping processes that will prevent you from representing distance in the tracing.
Continue tracing the neuronal processes in the current section 