IUR Planes Optical Fractionator

Use this probe to estimate the ratio of total line length within an Optical Fractionator sampling scheme.

The program displays sets of parallel lines over the material to be examined. These lines represent planes that extend along the Z axis. You will place markers where the lineal features of interest intersect the planes within each Optical Fractionator counting frame.

If you don't have isotropic sections, use Isotropic Virtual Planes or Spaceballs instead.

 

  • Thick IUR slices (i.e., isotropic sections)
  • Lineal structure to be sampled (e.g., fibers) with a very small cross-sectional area or a single one-dimensional lineal feature that can be identified (e.g., a medial axis)

See IUR Planes Optical Fractionator formulas

  1. Start the Serial Section Manager if multiple sections are to be sampled.
  2. At low magnification, trace a contour of the area of interest.
  3. At high magnification, click Probes>Tools>Define counting frame to define the frame.
  4. Click Probes>Tools>Preview SRS Layout to set the desired spacing between counting frames.
  5. Switch to a lens that is suitable for viewing the linear structures.
  6. Click Probes>All probes>Length>IUR Optical Fractionator to open the IUR Planes Fractionator dialog box. Adjust the settings and click OK.

    We recommend that you select Refocus to top of sectionat each grid site. This is the most accurate way to calculate section thickness.

    • Under Measure Thickness, if you have very uneven tissue thickness within each section, check Measure section thickness at each selected grid site.
      • If you use this option, thicknesses are recorded and used to report Number Weighted Section Thickness results, which take into account the varying thickness in calculating your final estimated total.
    • Spacing of Planes: Allow for 2 to 4 intersections between the planes and the lineal feature of interest per counting frame.
    • Angle of Sampling Planes: If no value is entered, the planes appear as horizontal lines. The planes always extend straight down through the section, so the lines do not appear to move as you focus through the section.
  7. The program randomly selects the starting point of the scan and moves the stage to the first counting frame with lines.
  8. The Focus Top of Section dialog box opens . Identify the top and click OK.
    • If you selected Measure section thickness at each selected grid site, the Focus Bottom of Section dialog box opens . Identify the bottom and click OK.
    • If the Z meter is enabled, it displays the disector's height in green . If you focus above or below the tissue (i.e., outside of the green area displayed in the Z meter), the cursor turns into .
  9. Select a marker for counting intersections.
  10. Click to mark wherever the planes (lines) cross the lineal features of interest within the counting frame. Mark intersections in all focal planes of the tissue that fall within the Z boundaries of the counting frame.
    • Count only lineal features that completely cross the displayed lines (or where the identifiable center of the lineal feature crosses the line).
    • Don't count cases in which the line is tangent to the lineal feature.
    • Count the intersection where the lineal feature is most clearly in focus.
    • It is OK to count a lineal feature twice if it intersects the same line twice within the counting frame.