Isotropic Fakir method

The Isotropic Fakir Method uses a linear probe consisting of three mutually orthogonal lines to estimate surface area in thick sections (slices). This probe can be used on sections cut in any orientation, as the probe lines themselves are isotropic in orientation. The number of intersections between the triplets of probe lines and the surface of the sampled object are counted, and surface area estimated from these counts.

This probe, as implemented in Stereo Investigator Cleared Tissue Edition, is not a fractionator probe. This means that the surface estimate is only valid for the sections sampled—if you skip sections, they are not taken into account. You may, however, keep track of the section fraction yourself as described by Kubínová (1998, section 3.2).

Thick tissue sections (slabs) in which multiple adjacent optical sections can be sampled.

  1. Optional: Use the Virtual Section Manager if you have serial sections.

    The probe doesn't take the reciprocal of the section sub-fraction and multiply by the estimate automatically; you need to perform this operation yourself.

  2. Click Probes>All probes>Surface>Isotropic Fakir Method.
  3. Enter the line separation ( Isotropic Lines Separation refers to the three sets of mutually orthogonal test lines).
    • Experiment to determine an appropriate line spacing so that a statistically significant number of intersections are achieved.
  4. Click OK. The program displays an array of parallel line segments.

    Each line-segment consists of a solid line and a dotted line; the border where the solid line turns into a dotted line describes a border-point. As you focus up and down, the line segment moves and the border between dotted and solid shifts. As a result, the combination of border-points will describe a line through the thickness of the tissue.

    This is a representation of a triplet of mutually orthogonal line segments. In the program, you only see the parallel line segments (there are 5 of these on the red probe-line); one side of a line segment will be dotted and the other side will be solid.

    As you focus in Z, the border-point on the line segments describes a vector through the thickness of the tissue (the red line segment that goes through the five shorter line-segments).

    Watch a video showing that the 3 vectors described by the shorter line segments are mutually orthogonal.

  5. Select a marker type.
  6. Place a marker wherever a line intersects the boundary of the particles or sampled region, where that boundary appears in the most clear focus.

    Turn your attention to where the solid line changes to a dotted line on the line-segments. If a border-point intersects a surface for which you are trying to estimate the density, mark it.

    Watch a video showing the three vectors of the triplet only. The small parallel line segments that describe the line (cf. previous illustration) are not visible.

  7. Focus up and down until all particle boundary intersections in the current field of view are marked.
  8. Click the marker to deselect it.
  9. Right-click, select Line Set 2 and switch to the second line orientation.
  10. Repeat steps 5–9 for all line sets.
  11. Once all line sets have been sampled, move to the next field of view of interest and sample all of the line sets in the new field.
  12. Repeat until all fields of interest have been sampled.
  13. Right-click and select Exit Isotropic Fakir to exit the probe.

 

See Isotropic Fakir formulas

Watch a demonstration: