11. View the sampling results

Procedure

View the results using one of the following methods:

Click a set in the Probe Runs list to select it and click the View Results button. The Sampling Results window is displayed.

Click Display Probe Run List (use after running more than one probe if you want results from multiple probe runs). The Previous Stereological Runs window is displayed.
1. From the list, highlight the probe runs of interest.
2. Click View Results. The Sampling Results window is displayed.
3. Click on a category in the left hand panel to display the corresponding results in the right hand panel.
  By default, the Sampling Results window initially displays the estimated population and marker total for the first marker type in the marker bar used for counting. Click on other marker types in the left hand panel to see the corresponding results in the right hand panel.
4. Optional: Click Export to export all results directly to Excel (2003 or later).

Interpretation

Population estimate results are generated for each individual marker type used for counting as well as for all markers combined.

Stereo Investigator provides four estimates and the results vary based on the type of average measurement used for the mounted (post-processing) section thickness. This is because the mounted section thickness value is divided by the disector height to calculate the height sampling fraction (hsf). See Optical Fractionator equation.

The average mounted section thickness can be determined before counting, or thickness measurements at each counting site can be recorded after selecting the Measure the mounted thickness while counting option in the Measure mounted thickness step of the Optical fractionator workflow. The number of measurements used to calculate the average 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).

We recommend that you report the estimate that best reflects the histological properties of the region of interest.

Estimated population using user-defined section thickness

Calculated using a single value entered manually for the average mounted (post-processing) section thickness measured before counting.

The average thickness may be entered manually in the Measure mounted thickness step of the Optical fractionator workflow under Manually enter the average mounted thickness.

If you don't enter a value for Manually enter the average mounted thickness in the Optical fractionator workflow, the estimate equals zero.
To change the thickness value after the counting procedure, click Edit Mounted Thickness in the Sampling results window, enter the desired thickness, and click OK to generate/update the Estimated population using user-defined 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.

Estimated population using mean section thickness

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

Calculated using the average of all the section thicknesses measured at each site during counting.

Because this estimate is generated from the mean of all obtained section thickness measurements, it is considered to be the most accurate estimate when measurements are performed at every site.

Estimated population using mean section thickness with counts

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

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

This estimate is a variation of (and in many cases nearly identical to) the Estimated population using mean section thickness.

Report this estimate if:

Sites without any cells do not contain a lot staining making it hard to decipher the top and bottom of the tissue correctly resulting in measurements that may not be accurate.
Measurements at some sites without any marked cells may not be accurate due to sites being located on or near damaged or folded tissue.

You chose to skip 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.

Estimated population using number weighted section thickness

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

Calculated using the average of only 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.

Report this estimate when tissue thickness was measured at every counting site and the thickness varies dramatically across the region(s) of interest (i.e., wavy tissue).

All estimates calculated from measurements obtained while counting (i.e., all but Estimated population using user-defined section thickness) should return similar results if the measurements were taken correctly.

Sampling results window

Displays the results of the probes selected in the Previous Stereological Runs window (see Display Probe Run List). Click on a category in the left hand panel to display the corresponding results in the right hand panel.

By default, the Sampling results window initially displays the estimated population and marker total for the first marker type in the marker bar used for counting. Click on other marker types in the left hand panel of the Sampling results window to see the corresponding results in the right hand panel.

Right panel parameters

The following results are displayed when the Parameters category is selected in the left hand panel of the Sampling Results window:

Data File Name: File name associated with the data set, if the data was already saved.
Date and Time: When the probe was completed.
Region: Name of the contour type that defines the region of interest. If runs from multiple contour types are selected, the contour name used for the first run is displayed.
Sampling Parameter Set:Name of the saved sampling parameter set (if one was used).
Number of Sampling Sites: Number of sampling sites visited on all selected sections.
Disector Height (Z) (µm): Z-axis height of each disector.
Disector Volume (XYZ) (µm³): Area of the disector based on the counting frame dimensions and the disector height.
Guard Zone Distance (µm): Z-axis height of the guard zones.
Contour Shape Factor:Shape factor provides information about the complexity of a contour and is calculated by dividing the perimeter of a contour by the square root of its area. Perimeter and area can be found in the contour measurement window.
Counting Frame Width (µm): X-axis width of each counting frame.
Counting Frame Height (µm): Y-axis height of each counting frame.
Counting Frame Area (µm²): Area of a single counting frame.
Sampling Grid (X) (µm): Distance between counting frames (sampling sites) along the X-axis.
Sampling Grid (Y) (µm): Distance between counting frames (sampling sites) along the Y-axis.
Sampling Grid Area (XY) (µm²): Area of each grid step (Sampling Grid Width x Sampling Grid Height).
Section Thickness (µm): Manually entered user-defined value for the post-processed or “mounted” section thickness. If no value is manually entered, this will be 0.00. Note, that obtaining population estimates using a manually entered section thickness in not recommended. We recommend measuring the section thickness at each site during counting.
Mean Measured Section Thickness (µm): Mean of all section thickness measurements recorded while counting.
Mean Measured Section Thickness with Counts (µm): Mean of section thickness measurements obtained at each counting site containing marked objects.
Number Weighted Mean Section Thickness (µm): Weighted mean of the section thickness measurements obtained at each counting site containing marked objects. The measurements are weighted by the number of objects associated with the corresponding site.
Section Evaluation Interval: The interval of the tissue sections used for counting. For example, if every third tissue section was counted, the Section Evaluation Interval is 3.

About export to Excel

Click Export to Excel in the Sampling Results window. Results of all selected probe runs are exported directly to Excel (2003 or later). Each category in left hand panel of the Sampling results window is exported to a separate worksheet within a single Excel file.

Explanation of Excel worksheets

Summary: Population estimate(s) and Coefficients of Error (CE) for each individual marker.
Parameters: File information and parameters associated with the selected probe runs. This information should be added to the Methods section of a publication to enable other researchers to test the reproducibility of the results.
Counts by Site: Lists 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 objects within a section or between sections.
Coefficient of Error: Lists all Coefficients of Error. Note: CEs will only be calculated if a minimum of three sections have been analyzed.
CE Variance Details: Raw variance data used in the Gundersen CE calculation. See Coefficients of Error.
Section Details: Lists marker counts by section.
All Markers Z Histogram and Individual Markers Z Histogram: Distribution of marked objects 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 objects marked in the middle of the histogram is probably due to an incomplete staining penetration.
Z Depth: Lists how many markers are contained in Z-depth bins of 1 micron each. These values are used to generate the Z depth histograms.
Z Depth Details: Lists the Z values for each marker. These values are used to generate the Z depth histograms.
Smoothness : Distribution of markers within your region of interest.
Planimetry : 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 : Z value of the sections and the actual Z at which the contours were drawn in each section.
Raw Report : Number of markers and tissue thickness per site.