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Calculated using a single value entered manually for the post-processed or “mounted” section thickness.

Because this estimate is generated with only one value for the section thickness, local variations in section thickness are not accounted for. As a result, this estimate should be considered the least accurate of the 4 available estimates. But if there is no section thickness variation (e.g., embedding protocols such as plastic embedding), reporting the Estimated population using user-defined section thickness is acceptable.

This value is typically entered manually, or calculated, in Step 5 of the Optical Fractionator workflow under the Manually enter the average mounted thickness method.

• If you don't enter a value for Manually enter the average mounted thickness, the estimate equals zero.
• To change the thickness value after the counting procedure, click the Edit Mounted Thickness button in the Sampling results window.

Calculated using the section thickness measurements recorded while counting.

These measurements are recorded in Step 5 of the Optical Fractionator workflow, after selecting Measure the mounted thickness while counting method.

The number of measurements used to calculate this estimate is based on the interval you entered (e.g., if you entered 2, you are prompted to set the top and bottom of the section at every other counting site). The measured thickness values from these sites are averaged to produce a mean measured thickness value used for the height sampling fraction calculation.

Because this estimate is generated from the mean of all obtained section thickness measurements, it is considered to be the most accurate estimate of the region of interest when measurements are not performed at every site.

You may also choose to use this value for low frequency events (e.g., Brdu+ neurons) with many counting sites containing zero objects when you want to generate an accurate average measured section thickness measurement for the hsf using a systematic interval for site measurement.

If you didn't measure the thickness of sections while counting, this estimate is not calculated.

This estimate is a variation of Estimated population using mean section thickness.

Calculated using only the section thickness measurements made at counting sites that contain marked objects (in other words, section thickness measurements from counting sites with NO counted objects aren't included in the calculated average).

These measurements are recorded in Step 5 of the Optical Fractionator workflow, after selecting Measure the mounted thickness while counting method.

In many cases, this estimate will be nearly identical to Estimated population using mean section thickness.

You may choose to use this value when :

• You chose to ignore measuring the section thickness where there were no objects.
• You made errors in section thickness measurement that were not corrected when there were no objects to be marked.

If you didn't measure the thickness of sections while counting, this estimate is not calculated.

Report this estimate when thickness was measured at every sampling site and when the section thickness varies dramatically across the sections that include the region of interest.

Calculated using only the section thickness measurements from counting sites that contain markers. These measured thickness values are then weighted by the number of objects associated with them to produce a weighted average.

The number weighted mean section thickness is reported in the Parameters section and is used to calculate the height sampling fraction.

 

 

 

Summary tab	Estimates and CEs for each individual marker.
Parameters tab	Information to be added to the Methods section of a publication to enable other researchers to test the reproducibility of the results.
Counts by Site tab	Information about the measured thickness and number of markers at each site. This information can be used to calculate your own CE or to compare thicknesses/number of cells within a section or between sections
Coefficient of Error tab	Several CEs.
CE Variance Details tab	Information related to the calculation of the Gundersen and Shaeffer CEs.
Section Details tab	Marker counts by section.
All Markers Z Histogram and Individual Markers Z Histogram tabs	Distribution of cells within the tissue. Ideally, with no sectioning artifact from the microtome blade, there is an equal number of markers placed at each “bin” from the top of the site. In practice, there are fewer markers at the bottom/top of each site; set your guard zones so that they cover these regions. A spike of cells at the top of the histogram could be caused by focusing through the tissue or counting cell bottoms at the top of the tissue as though they were cell tops (assuming that cell tops are the unique point you're trying to count). A lower number of cells marked in the middle of the histogram is probably due to an incomplete staining penetration.
Z Depth tab	Raw data necessary for your Z depth histogram.
Z Depth Details tab	Actual Z values for each marker (rather than markers per bin).
Smoothness tab	Distribution of markers within your ROI.
Planimetry tab	Area of each section and volume calculated from this area. Note that this information is biased; for an unbiased estimate of area/volume, use the Cavalieri Estimator.
Z Order tab	Z value of the sections and the actual Z at which the contour were drawn in each section.
Raw Report tab	Number of markers and tissue thickness per site.