Placing fiducial markers and contours

Purpose

What is a fiducial?

Fiducials are distinctive locations/structures on specimens that you mark as references/landmarks. Fiducials are often used to help align a data file with its corresponding experimental sample, which can be an image or a live camera feed, so that you can resume data collection. Fiducials are also used for serial section reconstruction.

Fiducial points can be marked by placing markers.

Alternatively, you can create a fiducial contour by tracing an accurate contour around a small region that can be easily found again.

Choosing fiducials

Fiducials should be easily visible at low magnification and unique enough to be easily found in an experimental sample at a later time. Examples:

  • If the tissue has a cut edge, a unique area along this line can be a fiducial.

  • Other examples of good fiducials include areas along the central canal of the spinal cord, the longitudinal fissure, or the aorta in transverse embryo sections, or even a unique grouping of cells.

  • For serial section reconstruction, any structure that runs longitudinally through the entire structure you have sectioned is a good fiducial.

Procedures

Placing a fiducial

We recommend that you first identify a fiducial point at low magnification and then adjust its placement at high magnification.

  1. At low magnification, identify a fiducial point.

  2. Place a marker at that location, or trace a contour around a small unique structure in the area.

  3. Switch to high magnification and adjust the placement of the marker or the shape of the contour to match the fiducial as accurately as possible:

    1. If you placed a fiducial marker, select and move the marker to align the center of the marker with the fiducial point.

    2. If you traced a fiducial contour, select the contour and move the points in the contour to match the shape of the fiducial region as accurately as possible.

Using a fiducial to align data files to samples

Placing a fiducial marker or contour on the first slide or first section in a series can be helpful for realigning a data file with a sample for continuation of data collection in future software sessions, or for data auditing. Using a single fiducial point helps ensure that any tracings or counting data for all the sections/slides will be aligned for the remainder of the session, and that any data added to the file will be aligned in future sessions. While accurate placement of the reference point can be sufficient for aligning data files, fiducial markers and contours can help refine the alignment. Fiducial contours in particular lend themselves to the most accurate alignment and would be the most useful for auditing of markers placed on counted cells.

  1. Open an existing data file containing a fiducial marker/contour.

  2. Place the slide containing the first section onto the microscope (or whichever slide you used to place the fiducial marker/contour).

  3. Place a reference point.

  4. At low magnification, use joy free to move the joystick to align the fiducial marker/contour to its corresponding fiducial point on the slide.

  5. Switch to high magnification.

  6. Still using joy free, refine the alignment of the marker/contour to match the location of the fiducial point exactly.

  7. Once the fiducials are aligned, exit joy free.

For counting data, fiducial markers/contours need to be placed when the data file is first created before any tracing or probe runs have been performed.

Using fiducials to aid in serial section reconstruction

Using fiducial markers or contours can be a useful tool when performing serial section reconstruction live on the microscope, or serial section reconstruction on a previously acquired series of images. Placing fiducial markers/contours in all sections of a structure you are reconstructing can help by giving a starting point for alignment as you move through the sections. If there is no structure present in all sections, as is often the case, we recommend marking fiducial points that are also found on the next section. As a result, each section is matched with the tracing of the preceding tracing, rather than with the first tracing.

Note that the alignment of sections is a bit of a subjective process. Fiducial markers/contours are a useful tool to help with aligning sections as accurately as possible.