I have completed counting the cells with optical fractionator, however when I click on the ‘view results’ the ‘estimated value using mean section thickness’ is showing 0 or single digit numbers while total markers counted is 500-900, and Gunderson error (m=1) is around 0.04 to 0.09. Kindly help me with this.
This issue is probably caused by the numbering format in your Excel,
e.g. Here in Brazil the numbering is “.” for Thousand and “,” for Decimal, however in English it is the inverse, this causes the value as 0.
Double click on the cell where the number is placed and look at excel top bar if the value appears
In addition to what Patrick mentioned, the most common cause of getting 0 in the “Estimated value using mean section thickness” is that in Step 5 of the Optical Fractionator Workflow “Measure mounted thickness at sampling sites” is deselected and a user defined thickness is entered instead. With these selections the software would not prompt you to measure tissue thickness while counting. You could have also selected “do not measure” at every site when prompted to measure the bottom of the tissue while counting.
Though this adds an extra step, measuring tissue thickness while counting is the most accurate way to obtain the height subfraction (hsf = section mounted thickness/counting frame height) in the Optical Fractionator formula as mounted tissue thickness can vary between sections of the same animal and tissue processing methods. Let’s say, for example, that you took a few measurements of tissue thickness from one animal and determined your mounted tissue thickness was 30um. However, for the second animal the tissue was allowed to dry out more than the others and the actual mounted thickness for that animal is 25um. If you decided to use 30um as your mounted thickness for all animals, you would end up overestimating the cell population in the second animal whose mounted thickness was actually 25um.
If you find that there is not much variance to the tissue thickness measurements you obtain while counting, you can reduce the interval at which you measure to make this process more efficient.
If you are measuring tissue thickness while counting and getting a 0 for the “Estimated value using mean section thickness”, I would suggest you open a support case so our specialists can help you with this inquiry.