Using Optical Fractionator to count offline after an SRS Image Stack acquisition

• Subject Information: Type your name, the subject of the study, and any notes pertaining to this study or animal.

• Use Saved Sampling Parameters: Select No.

• Enter Serial Section information: The Section’s Cut Thickness used in the SRS workflow is displayed.

  • To modify the thickness, click the Edit Serial Sections button.

• Measure the mounted thickness while counting is recommended at every sampling site (or at least an evaluation interval) to ensure an unbiased estimation.

• If you select Manually enter the average mounted thickness, enter the Average Mounted Thickness, under Manual Adjustment.

• If you select Measure mounted thickness before counting, click the Start Taking Measurements button below to measure the section thickness at several sites prior to counting.

If you only collected images for the disector height, enter the section thickness after shrinkage manually. Keep in mind that, if you enter an incorrect thickness value, you are introducing sampling error and bias to the estimation.

• If you set the Probe Height and Guard Zones in the Acquire SRS Image Stack Workflow (step 8: Image Stack Settings), the correct values for the Top Guard Zone Height and Optical Disector Height are displayed.

• If you didn't enter the Top Guard Zone Height and the Optical Disector Height values during the SRS Image Stack acquisition, enter them now.

• Under Focus Method, select Manual Focus.

1. Under Regions of Interest, expand Section 1 by clicking the + sign, expand the region of interest by clicking the + sign, and highlight Acquired Stacks.

2. Click the Play button to start counting. The stack is loaded and you are directed to the first sampling site.

If you changed the sampling parameters between the SRS workflow and Optical Fractionator workflow: Select Use Current Settings and click OK in the Choose Probe Configuration window.


3. The Focus Top of Section dialog box is displayed. Using the PageUp key, focus to the top of the stack, then click OK in the dialog box.

4. If applicable, the Focus Bottom of Section dialog box is displayed. Using the PageDown key, focus to the bottom of the stack, then click OK.

5. Select a marker from the Use Marker drop-down menu in the workflow panel, or from the marker toolbar.

6. Count the cells following the counting rules.

7. Click the fast forward button to go to the next site.

8. Repeat step 4.a–4.g for all the sections.

9. Click the I’m Finished Counting button when done.

10. Click the Next Step button to move to Step 5.

1. Click the Probe Run List button.

2. The Previous Stereological Runs window is displayed. Select all relevant sections and click the View Results button.

3. The Sampling Results window is displayed.

   • Parameters for your study appear first.

   • To see the estimated total cell numbers, highlight the marker name on the left—Do not be alarmed if some of the results are 0.

     • Estimated Population using User Defined Section Thickness: The estimate is calculated using the manual thickness entered prior to counting the cells.

       • Use this number ONLY if you have measured the thickness separately, before counting ( see Measuring thickness)

       • Equals zero if you didn't enter the thickness value manually.

     • Estimated Population using Mean Section Thickness: The estimate is calculated based on the average thickness of the tissue. We refer to section thickness that you measured within the workflow at every site, whether cells were counted or not.

     • Estimated Population using Mean Section Thickness (only using sites with counts): The estimate is calculated based on the average thickness of the tissue. We refer to the section thickness that you measured within the workflow only for the sites where you marked cells.

     • Estimated Population using Number Weighted Section Thickness: The estimate takes into account wavy tissue.

       • Use this number if the section thickness varies between sampling sites.

   To change the section thickness, click the Edit Cut Section Thickness button in the Sampling Results window and enter the correct thickness after shrinkage.

   • To display the CEs, click their names on the left.

   • To view planimetry results, click Planimetry on the left. The results report the area and volume of the region of interest. They are correct only if the contours used to define the region of interest were drawn accurately.

1. Go to File>Preferences and select the Images panel.

2. Turn off (uncheck) Zoom to fit on load.

3. Turn on (check) Remove off-screen image stacks from memory.

Most users combine SRS image stacks with the Optical Fractionator to count cells (see Using Optical Fractionator to count offline after an SRS Image Stack acquisition ).

When counting:

1. Go to File>Preferences and select the Movement panel.

2. Turn on (check) focus with mouse wheel and set the Z distance per wheel click equal to the distance between images in your image stack. This will allow you to focus with the mouse wheel instead of the page up/page down keys.