Fractionator

Purpose

Number

Mono-layer

The fractionator probe is most often used to estimate population counts of 2D specimens such as plated cells for tissue culture analysis. It is typically employed to sample populations that are too large to count exhaustively.

Procedure

Setting up to use the fractionator probe

Working with an SRS image series acquired previously

Follow the detailed instructions here; an abbreviated version is shown below:

  1. Open the data file for an SRS image or image-stack series.

  2. Click Select Acquired SRS Image Series from the Tools section of the Probes ribbon.

  3. Choose the image series and probe, then click OK.

    The Fractionator probe opens.

  4. Count objects as described below.

Working with a live-camera feed

  1. Place a reference point; the point can be placed arbitrarily or at a particular location on the slide.
  2. Select a low magnification lens that allows for the efficient and accurate definition of the region of interest (e.g., 4x).
  3. Trace a region of interest using the contour tool.
  4. Switch to an objective and lens that allow you to accurately identify the cells to be counted (e.g., 40x, or 20x for a sparse population).
  5. Click Probes>Define Counting Frame to set the size of the Counting Frame.

    Size the counting frame so that it contains a number of cells that you can comfortably mark without errors (i.e., no double count or missing cell).

  6. Use Probes>Preview SRS Layout to define and preview the Systematic Random Sampling (SRS) grid. Click anywhere in the window to exit the preview.
  7. Click the Fractionator button in the Number drop-down menu on the Probes ribbon to start Fractionator.
    1. You'll be prompted to open the Serial Section Manager. Click No.
    2. The Fractionator dialog box appears, displaying the previously determined counting frame size and grid size. Click OK to move the stage to the first sampling site (the first site is determined randomly).

Counting objects

  1. Select a marker from the Markers toolbar and mark all the objects of interest included in the counting frame following the counting rules.

    2. If the objects are double- or tripled-labeled, use combination markers to count the different cell types. See Colocalize markers .

  2. When finished marking, press F2 (or right-click and select Next site) to move to the next site.

    When finished with all the sites, the probe ends.

    Optional: if you have multiple regions, move to the next region and repeat with appropriate sampling parameters for the region.

  3. To view the results, click Probes>Probe run list.

    1. Select the Fraction probe for each contour (each contour represents 1 cover slip).

    2. Click View Results. In the Sampling Results window:

      • Click Counts by site for number of markersfor each site visited.
      • Click CE Scheaffer or CE Schmitz-Hof to view the coefficient of errors in the right panel; the values are more applicable to cell estimations in tissue sections than on coverslips. The CE can give you an indication of the homogeneity of the cellular distribution on the coverslip (see Coefficients of Error).

      • Click Planimetry to see the area of the coverslip that can be used to calculate cell density (value in the right panel).

        If you don't see Planimetry, go to File>Preferences>Stereology Preferences>Results>Volume estimations and check the box to Display Planimetric Results.

To end a Fractionator probe before it is completed, click the Fractionator button again at any time during the scan.

 

See Fractionator formulas