5. Measure mounted thickness

Overview

The calculations for Optical Fractionator require the measured thickness of the tissue post processing. We recommend that you measure the tissue thickness at every sampling site for the most accurate cell estimates. The measured thickness from the counting sites is averaged and used in the calculations of the height sampling fraction.

Since most studies require the option to Measure the mounted thickness while counting, it is selected by default, along with the option to Refocus to top of section at each grid site, which is used to determine the top of the guard zone.

Bear in mind that one method of calculating the population estimate — Population estimate by number-weighted section thickness— requires a thickness measurement at each site. Also note that the Resample Disector tool (designed to help you determine the guard zone extent), requires a thickness measurement at each site.

Choose Your Method(s) for Measuring

Check the box to select one of the following methods for measuring:

Measure the mounted thickness while counting

Evaluation Interval: set to "1" by default.

If thickness is uniform across sections, increasing the Evaluation Interval will save some time, but we still recommend measuring the thickness at every site to increase the accuracy of the estimates.

For some studies, researchers may choose to measure the section thickness at an interval, for example, every 3rd sampling site (i.e., Evaluation Interval=3), to save time. It is acceptable if the thickness of the sections is uniform.

Manually enter the average mounted thickness

Use this option if you know the average mounted thickness (for example, you are counting from sections that tend to have a uniform thickness (e.g., celloidin) , or something that shrinks uniformly, and you are satisfied that you have adequately measured it).

This will save time while counting, but can result in inaccurate estimates especially if your tissue is wavy.

Measure mounted thickness before counting

Use for accurate measurements using the top and the bottom of each section.

Select this option if, when starting a new study, you have not pre-measured the thickness (see Manually enter the average mounted thickness above).

Take a few measurements of your tissue thickness at this step:

Click Start Taking Measurements.
Using the joystick, focus on the top of a section; Click Set Top of Section.
Focus on the bottom of the section and click Set Bottom of Section.
Click Add Measurement to List.
Continue with steps 2 through 4 (5 to 10 measures should give you a representative measurement).
Use Revisit Measurement to verify and measure again, or Delete Measurement if you have made a mistake.
Click Stop Taking Measurements when finished.

You’ll need to know the approximate thickness of your tissue when you define the height of your disector and guard zones (described in 8. Define disector options).