Optical fractionator workflow

Purpose

The Optical fractionator probe is used to perform a systematic random sampling of a predefined 3D region of interest and estimating the total number of specific objects in the specimen, for example the number of cells.

The Optical fractionator workflow steps you through the process of setting up to use the probe and mark/count your objects of interest.

More information about the optical fractionator is available at our stereology information website.

See also our webinar (YouTube, 50:33): Unbiased stereology to determine the number of cells in a region of interest

Starting the optical fractionator workflow

  1. Click the Optical Fractionator workflow button on the Probes ribbon.

    If you haven't used the workflow recently, find the Optical Fractionator workflow button in the Number drop-down menu in the All Probes section of the Probes ribbon.

  2. In the dialog box that opens, the following choices may be available, depending on what files, if any are currently open. Choose what you would like to do:

    • Start a new subject: Count objects on a live-camera feed from your microscope or in an open image file that contains the entire region to be counted.
    • Continue working with this subject: Continue working in an open data file.
    • Load subject data from existing file: Load an MBF Bioscience data file that is not currently open.
    • Start a new subject from image stacks:Load image stacks acquired in a systematic random sampling (SRS) manner acquired using another microscope system (i.e., not an MBF Bioscience system).

Optical fractionator workflow steps

The workflow is dynamic; your choices determine which steps are included, and what information is requested and displayed. Because of this, the step numbers you see onscreen in Stereo Investigator Whole Slide Edition may be different than those in the complete workflow described here.

Click any of the underlined steps to jump to the instructions:

Indicate the Areas Used for Counting

Procedure

2. Set microscope to low magnification

Procedure

Procedure

Define Probe Configuration

5. Measure mounted thickness

Procedure

Overview

8. Define disector options

Procedure

Perform Counting

Procedure

Procedure

Commands available in every step of the workflow:

New workflow: Click the new workflow button to start over; the settings will revert to the defaults or those specified in the previous completed workflow.
Previous step / Next Step: Click to advance in the workflow or revisit a previous step. Alternatively, you can click the steps listed at the top of the workflow to jump to that step.
Click the help link to view information in the User Guide on completing the current step in the workflow.

About the Optical Fractionator

The Optical Fractionator (West, et al., 1991) combines the Optical Disector and the Fractionator for counting. It is not affected by tissue shrinkage and does not require rigorous definitions of structural boundaries.

Using the Optical Fractionator involves counting objects with optical disectors in a uniform systematic sample that constitutes a known fraction of the volume of the region being analyzed. In practice, this is accomplished by systematically sampling a known fraction of the section thickness, sectional area, or number of sections that contain the region of interest.

Use the Optical Fractionator to perform systematic sampling of objects distributed within a series of serial sections to estimate their population number in a volume. Properly designed systematic sampling yields unbiased estimates of population number.

The theory underlying this sampling methodology also makes it possible to estimate the precision of the population size estimate for a single subject; this estimate of precision is called the Coefficient of Error (CE).

 

See also Optical fractionator formulas.