Measure mounted thickness
Optical Fractionator - step 5
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 will require the Measure the mounted thickness while counting option, this option is selected by default, along with 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
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.
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 you have measured it enough).
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 have an approximation of your tissue thickness when you define the height of your disector and guard zones (described in Step 8).