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Area Fraction Fractionator Probe

Area & Volume

  

The Area Fraction Fractionator (AFF) is essentially a Cavalieri point counting estimate of area and volume performed on a systematically selected fraction of the tissue. The probe is designed to estimate the fraction of a region occupied by a sub-region. Any number of sub-regions may be examined using the AFF. This area fraction probe is implemented by performing a fractionator scan of the area of interest, while overlaying a grid of points (as in the Cavalieri estimator).

As with the Cavalieri probe, a shortcoming of the method is that there may be uncertainty in judging whether points near the boundaries of the area are inside or outside of the region of interest or object of interest.

Examples

  • Compare the ratio of white matter to gray matter in the spinal cord.
  • Quantify the ratio of muscle fiber to connective tissue to vasculature in a muscle.

Thin/Thick

  

Isotropic/Vertical/Preferential sections

  
ClosedPrerequisites & design

The design of the Area Fraction Fractionator probe includes:

  • The size of the counting frame.
  • The distance between fractionator sampling sites.
  • The appropriate distance between the points of the grid that will be used.

You need to know the interval between the sections to be sampled.

Here are some general guidelines for designing an area fraction probe:

  • The fractionator scan grid size should be set to sample an appropriate number of sites per section to achieve the required precision. The distribution of the objects of interest will dictate the grid size. Larger grid sizes will save time, but the grid size must be small enough that it will not skip over any important region of the tissue.
  • The counting frame should be sized so that it is large enough to distinguish between the regions of interest within the counting frame. A general rule of thumb is that the counting frame is 1/3-2/3 of the field of view used for viewing the objects of interest.
  • The smaller the distance between lattice grid points, the more accurate the estimate. However, using too many points can be inefficient because it increases the time spent on each site.

 

ClosedProcedure

Use the Options>General Preferences>AutoSave tab to set your AutoSave settings to minutes instead of number of data points.

  1. Place a reference point.
  2. Define your sections with the Serial Section Manager. The Section Cut Thickness is used to calculate the estimates.
  3. Trace the region(s) of interest at low magnification (i.e., with low precision). Tracing contours
  4. Select a lens suitable to mark the tissue.
  5. Use Probes>Define Counting Frame.
  6. Use Probes>Preview SRS Layout. Also Determining Sampling Precision
  7. Select Probes>Area Fraction Fractionator to open the Area Fraction window.
    1. The counting frame size and the XY placement of the grid you specified in Step 5 are entered.
      • Pick a grid spacing for the distance between points. The grid can also be rotated randomly. 
    2. Select marker types:
      • Left Button Marker (Left Button refers to the left mouse button): Used to mark single points or groups of adjacent points for the sub-region (e.g., plaque, lesion). You will mark the grid points that meet these 2 criteria: 1) they appear on the objects or region of interest, and 2) they are contained by the counting frame.
      • Middle Button Marker (Middle Button refers to the mouse wheel button): Used to fill the region of interest, that is, area of the contour overlaid by the counting frame (e.g., cortex). You will mark the grid points that meet all 3 criteria: 1) they appear in the region of interest, 2) they are contained within the counting frame, and 3) they do not appear on the objects of interest.
  8. Mark areas of interest—and adjust the size of your cursor with the mouse wheel if necessary:
    • To fill the entire area inside the contour with Middle Button Markers (cf. Step 7), click the mouse wheel button.
    • To mark a single point, left-click once.
    • To mark several adjacent points, drag the mouse (Paint mode).
    ClosedAlternatively, use the Marquee mode to mark more efficiently
    1. Right-click and select Marquee Mode.
    2. Drag the mouse across the area of interest; a white rectangle is drawn. All points within that rectangle are marked.

    To measure the area fraction of several types of objects, use additional marker types -- one marker type for each region of interest.
    Select a marker from the Marker toolbar then place markers.
    Repeat with a different marker for each region of interest.

    ClosedAll right-click menu options

    Next scan site -- Advances you to the next site to mark points when you are done at the current site.

    Previous scan site -- Returns you to the most recent site where you marked points.

    Snap to grid mode -- Any marker you place appears on the nearest point. Required to use Paint mode, Erase mode, Marquee mode, Fill mode, Replace mode.

    Paint mode -- Use to mark groups of adjacent points.

    Erase mode -- Use to erase markers.

    Marquee mode -- Places markers within a rectangle.

    Fill mode -- Fill in all points inside of a previously traced closed contours.

    Replace mode -- Replaces one marker with another.

    Preview area fraction -- Provides a bird's-eye view of all markers.

  9. When you are finished marking, right-click and select Next Scan Site.

    To return to the previous site, right-click and select Previous Scan Site.

  10. Repeat the Steps 8 and 9 until all sites have been visited.
  11. If you have multiple sections, move to the next section and repeat Steps 8–12.
  12. To view the results, use Probes>Display Probe Run List.
    • Area Fraction: Fraction of tissue (region) occupied by the sub-region (plaques)
    • Estimated Area: Area of tissue (region) occupied by the sub-region (plaques)
    • Planimetry: Area and volume of the region of interest based on the traced contour.
ClosedDetermining a CE

We recommend that you use this Excel spreadsheet to generate a CE.

Follow the instructions below to use the spreadsheet:

  • Use the Counts by site values displayed in the Sampling Results dialog box .
  • Update the formulas if more than 10 sections were used.
  • Shape factor: Average shape factor for all sections. The default is 4; you may use a different value. Shape Factor for details on calculating the shape factor.
  • Variance (Gundersen): The default is m=1 and the divider value is 240; if m=0, set the divider value to 12 instead of 240.

 

 

See Equations: Area Fraction Fractionator

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