Optical Fractionator

The size of the counting frame should be small enough to fit in a single field of view when using the objective lens to count cells. We recommend that you size the counting frame so that it includes only a few objects.

The thickness of the counting frame, added to the thickness of the upper and lower guard zones, must be less than or equal to the minimum physical section thickness.

Counting Frame Thickness + Guard Zones Thickness <= Min Physical Section Thickness

Thus, the maximum thickness of the counting frame is constrained by the section thickness (and guard zone thickness), while the minimum counting frame thickness is determined by the cell distribution and the depth of field of the objective lens that will be used to count the cells. This latter factor is important since only cells that come into focus while focusing down through the counting frame will be counted. This means that the depth of field of the objective lens should be significantly less than the thickness of the counting frame.

The guard zones should normally be thick enough to ensure that the tissue sampled by the 3D counting frame is not disturbed by the process of preparing the sections.

The minimum guard zone thickness required depends on how the tissue was sectioned, and on how it was processed before and after sectioning (e.g., guard zones may be unnecessary for plastic-embedded sections).

See Guard Zones

The height of the optical disector needs to be viewed in light of several considerations:

• The thickness of the optical disector must be less than the thinnest portion of any section.
• It must be thick enough to provide a representative volume.
• It must be significantly thicker than the depth of field for the objective lens that is used when counting cells.

If sections were cut at 40 microns and the final section thickness varies between 18 and 20 microns, the optical disector height could be 12 microns, allowing the top and bottom guard zones to be 3 microns.

There are several methods of determining an appropriate section increment (see Determining Sampling Precision). This value is constrained by the total number of sections in the series that contain some portion of the volume of interest. Sample enough sections so that what you see is a representative cross-sectional volume of the region of interest.

Normally, you should neither sample every section, nor only one or two sections.

It is important to know the minimum actual section thickness (mounted thickness) in order to properly determine the thickness of the counting frames and their guard zones. Most histological processes result in shrinkage along the Z-axis of a section (due to greater exposed surface area). Some cutting procedures result in inconsistent section thickness. Even if a section starts out with a consistent section thickness, different areas within the section can shrink differently, depending on the characteristics of the tissue.

To measure the section thickness:

1. Click the Z Meter icon to display the Z Meter.
2. Focus to the top of a section using a lens that has a depth of field significantly smaller than the section thickness.
   • If you can't focus on the top of the section, try to focus on the first object below the top of the section.
   • If there is no object at the top of the section, focus on the extracellular matrix at the top of the section. This can be difficult to execute, but it is essential for an accurate determination of section thickness.
3. Press ALT+S. Stereo Investigator sets the Z position to 0.
4. Focus to the bottom of the section.
   • If you cannot identify the bottom of the section, try to adjust the stage height such that the bottom of the lowest object in the field of view is just leaving focus as you are focusing down through the section.
5. Look at the Z (focal) depth value (third of the three coordinate numbers displayed in the parentheses) in the lower-left of the status pane . This value is the distance (in microns) between the top and the bottom focal planes you have chosen. If you focused at the top and bottom of the section, it is the section thickness at this particular location.

Alternatively, you can measure the section thickness during step 5 of the Optical Fractionator Workflow.

Stereo Investigator

The optimal mounted thickness is 20 to 30 microns; this allows for an optical disector with four to five distinct optical planes. Determining the optimal thickness is important and must be done at the pilot study stage to obtain the best results.

If you have a very high numerical aperture 100X oil lens, you can expect to have distinct optical planes every 0.5–0.7 microns. At lower magnification or with lower NA lenses, the optical sections (depth of field) will be thicker.

If sections are too thin, there may not be enough room for adequate guard zones, or the objective lens to be used may not have an adequately thin depth of field for this protocol. If sections are too thick, it may be very hard to focus clearly to a given depth due to visibility constraints, working distances of objective lenses, etc. There may also be issues of consistent staining throughout the section thickness.

Once the section increment has been determined, the starting section index should be chosen at random. Stereo Investigator lets you do this easily when you define the first section of a series.

1. Place a representative slide under the microscope and navigate to the region of interest using a low magnification lens (remember to select this lens from the drop-down menu in the toolbar).
2. Before tracing the region of interest, define your sections with the Serial Section Manager.
3. Draw a contour around the region of interest in which you want to count cells.
4. Switch to a high power objective lens suitable for counting cells; remember to select this lens from the drop-down menu in the toolbar.
5. Use Define Counting Frame.
6. Select a marker for counting cells. To count multiple types of cells, use a different marker type for each cell type.

1. Click Probes>Optical Fractionator. The Optical Fractionator window opens .
   • We recommend that you select Refocus to top of section at each grid site. This is the most accurate way to calculate section thickness.
   • Under Measure Thickness, if you have very uneven tissue thickness within each section, check Measure section thickness at each selected grid site.
     • If you use this option, thicknesses are recorded and used to report Number Weighted Section Thickness results, which take into account the varying thickness in calculating your final estimated total.
2. Click OK. Stereo Investigator randomly selects the starting point of the scan and moves the stage to the first counting frame. A random starting point is necessary to ensure unbiased sampling.
3. The Focus Top of Section dialog box opens . Identify the top and click OK.
   • If you selected Measure section thickness at each selected grid site, the Focus Bottom of Section dialog box opens . Identify the bottom and click OK.
   • If the Z meter is enabled, it displays the disector's height in green . If you focus above or below the tissue (i.e., outside of the green area displayed in the Z meter), the cursor turns into .
4. Stereo Investigator automatically moves the stage to the top of the counting frame.
5. Count cells within the 3D counting frame by placing markers. See Counting rules for the counting frame
6. Once all the cells have been counted, right-click and select Next Scan Site button to move to the next site.
   
7. Repeat steps 3–7 until you have examined all of the counting frames distributed across the region of interest for this section.

1. Select the section in the Serial Section Manager.
2. Select a low magnification lens (remember to select this lens from the drop-down menu in the toolbar).
3. Draw a contour around the region of interest in which you want to count cells.
4. Switch to a high power objective lens suitable for counting cells (remember to select this lens from the drop-down menu in the toolbar).
5. Click Probes>Optical Fractionator and run Optical Fractionator again.

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.