Tracing Neurons in Serial Sections
Before you start...
- Use all the sections containing the neuron(s) of interest.
- Determine the orientation of the sections.
- Determine the tracing method best suited to your tissue: While many users start tracing from the cell body, some prefer to work in one direction, starting at the furthest extent of a cell's processes.
- For best results, trace all of the stained material in a single section before moving on to the next section. This avoids the small alignment errors found upon returning to a section multiple times.
- When using live images, be knowledgeable about the concepts of moving the stage and aligning serial sections. Aligning Serial Sections from a Live Image
- Use the relative depth filter in the Orthogonal View panel to make neuronal tracing easier. Orthogonal View
- Set for a range lower than the section thickness, but higher than a single focal plan.
- If the range is too low, processes that move up and down in Z appear fragmented, which can be confusing.
If your system is not configured with a focus position encoder and doesn't have an internal Z motor, focus adjustments made directly on the coarse focus of the microscope are not recorded and result in a loss of Z-axis data.
Procedure
This procedure uses the top of each consecutive section to set the Z depth of the starting points.
- Tracing regions of interest/fiducial contours
- Tracing neuronal processes in the first section
- Moving to the subsequent section and aligning tissue and tracing from the first section
- Tracing the neuronal processes in the next section
Refining the tracing