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

If you are counting after acquiring image stacks using the SRS image-stack series workflow in Stereo Investigator, see Using Optical Fractionator to count offline after an SRS Image Stack acquisition .

If you are counting image stacks acquired in a systematic random sampling (SRS) manner using another microscope system (i.e., not an MBF Bioscience system), see Using Optical Fractionator with external image sources .

Requirements

  • Thick sections (significantly thicker than the diameter of the tubules/fibers to be measured)
  • Structure of interest that is stained and visible through the depth of the tissue section.
  • Thin focal planes achieved with a high magnification, high numerical aperture lens, generally oil immersion.
  • Approximate section thickness: Use a high power objective lens with a high numerical aperture to focus at the top and bottom of a few sites throughout your sections to get a rough idea of the section thickness. This value can be edited later, but an approximate value is needed for setting up the disector and guard zones.

We recommended that you place a reference point in an easy to find and recognizable location before starting this probe workflow. When working with a live camera feed, the placement of the reference point is essential for maintaining accurate alignment between a data file and its corresponding experimental sample when collecting data over multiple sessions. If a reference point has not been placed before opening a workflow, Stereo Investigator will automatically place it in the top left corner of the Main window. This location may not be ideal for resuming data collection in subsequent sessions.

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 using another microscope system (i.e., not an MBF Bioscience system). See Using Optical Fractionator with external image sources .

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 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

1. Set up the subject

2. Set microscope to low magnification

3. Trace your region(s) of interest

4. Set microscope to high magnification

Define Probe Configuration

5. Measure mounted thickness

6. Define the counting frame size

7. Define SRS grid layout

8. Define disector options

9. Save sampling parameters

Perform Counting

10. Count objects

11. View the sampling results

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.