L-Cycloid Optical Fractionator

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

The program displays sets of curves (cycloids) in a grid pattern over the objects of interest. You will place markers where the cycloids cross the projected lineal features of interest within the Optical Fractionator counting frames.

 

  • Vertical Uniform Random sections, or vertical slices of constant thickness having a random rotation around vertical axis
  • identifiable vertical axis visible in every section
  • lineal structure with a very small cross-sectional area or a single one-dimensional lineal feature that can be identified (e.g., a medial axis)

See L-Cycloid Optical Fractionator formulas

  1. At low magnification, trace a contour of the area of interest.
  2. Switch to a high magnification lens suitable for viewing the linear structures.
  3. Click Probes>All probes>Length>L-Cycloid Optical Fractionator. 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.
    • Cycloid Width: Enter a value that allows for 2-4 intersections between the cycloid and the lineal feature of interest for each counting frame.
  4. Click OK. The program randomly selects the starting point of the scan and moves the stage to the first counting frame with cycloids.
  5. 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 .
  6. Right-click and select Define Vertical Axis.
    1. Drag the head of the arrow to align its vertical axis with the vertical axis of the tissue (if you can't determine the vertical axis at high magnification, switch to a low magnification lens).
    2. Right-click and select Accept.
  7. Select a marker for counting intersections
  8. Click to mark each point where the cycloid curves 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.
  9. When all intersections in a counting frame have been marked, right-click and select Next Scan Site to move to the next site.
  10. To view results, click Probes>Tools>Probe run list.