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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

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.

• 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.

   

Procedure

This procedure uses the top of each consecutive section to set the Z depth of the starting points.

1. Tracing regions of interest/fiducial contours
2. Tracing neuronal processes in the first section
3. Moving to the subsequent section and aligning tissue and tracing from the first section
4. Tracing the neuronal processes in the next section

Refining the tracing

• Splicing process fragments

Microlucida 3 | MBF Bioscience Support Center | Downloads